CN103800906A - Compositions for stimulation of mammalian innate immune resistance to pathogens - Google Patents

Abstract

Embodiments of the invention are directed to methods of treating, inhibiting or attenuating a microbial infection in an individual who has or is at risk for developing such an infection, comprising the step of administering an effective amount of a TLR9 agonist and a TLR 2/6 agonist to the individual.

Description

For stimulating the compositions of the innate immunity resistance of mammal to pathogen

The application is the divisional application of the application that application number is 201080022634.7, denomination of invention is " for stimulating the compositions of the innate immunity resistance of mammal to pathogen ", and this mother's case application is the application that the PCT application PCT/US2010/028658 that submits on March 25th, 2010 enters the China national stage.

Background of invention

I. invention field

Relate generally to microbiology of the present invention, immunology and antimicrobial agents therapeutics field.Particularly, the compositions and methods of the invention relate to and use small molecule compositions to regulate the innate immunity in individual lung, for treatment or alleviate infected by microbes or intrusion.

II. background of invention

Lung comes from the structural requirement of air exchange to the susceptibility infecting.In order to support ventilation, people is constantly by 100m ²lung surface area exposure is in external environment.Lung is not only exposed to air, is also exposed in the granule, droplet and the pathogen that wherein suspended.From be wrapped in the skin surface in obstructed transdermal or there is the gastrointestinal tract of thick absorption rete malpighii different, lung presents the overall situation interface with minimum barrier defence.The demand of gas diffusion without prejudice forecloses larger barrier.

Although its fragile structure, lung generally can successfully defend to infect (Knowles etc., 2002 by multiple machinery, body fluid and cell mechanism; Martin and Frevert, 2005; Rogan, etc., 2006; Travis, etc., 2001); (Mizgerd, 2008; Bals and Hiemstra, 2004; Bartlett etc., 2008; Hiemstra, 2007; Hippenstiel etc., 2006; Schutte and McCray, 2002).The microbial pathogens that great majority are inhaled into can not penetrate into alveolar owing to being embedded in airway walls, they are because mucus is trapped in airway walls, then discharge (Knowles etc., 2002) by mucociliary elevator system (mucociliary escalator system).For those pathogen of escaping this destiny, in air flue inwall fluid the composing type of antimicrobial peptide exist limited they growth (Rogan, etc., 2006; Travis, etc., 2001).The pulmonary alveolar macrophage that is positioned at air chamber distal-most end can absorb these organisms, clears up thus lung to prevent possible infection.

Although be often considered to the barrier of passive gas exchange, air flue and alveolar epithelium in the time running into pathogen and stimulate by there is great partial structurtes and changing function for the defence of basic pulmonary provides supplementary.Respond to virus, fungus or alterative inflammation, Airway secretion cell improves rapidly its height and be filled with secreted granule in its apical cell's matter, this process is called raw (inflammatory metaplasia) (Evans etc., 2004 of inflammatoryization; Williams etc., 2006).Under pathogen exists, alveolar epithelium activates its plasma membrane system and secretion machine, makes thus leukocyte participate in pulmonary's protection (Evans etc., 2005).May the most important thing is, with the microbial interaction of airway epithelial pattern recognition receptors, many microbial products be expressed in air flue inwall fluid, comprise alexin, cathelicidins, lysozyme and reactive oxygen species (Rogan etc., 2006; Forteza etc., 2005; Akinbi etc., 2000; Bals and Hiemstra, 2004; Bals and Hiemstra, 2006).It should be noted, pneumonia (bacteroidal or viral) is the lethal first cause of infection in worldwide.

Exist suppressing and/or the treatment additive method of infected by microbes and the needs of compositions.

Summary of the invention

The invention provides and stimulate the compositions of congenital resistance (be excited congenital resistance (Stimulated Innate Resistance, StIR)) and use such composition to stimulate the method for StIR.In certain embodiments, StIR is the StIR of lung.An aspect of of the present present invention provides higher treatment/toxicity ratio or index.Embodiments more of the present invention for example comprise, for strengthening for example, compositions, preparation and method to the biophylaxis of infecting (experimenter is to the immunity infecting) of mammal (people) experimenter.In some aspects, compositions of the present invention is deposited in individual lung with effective dose.Aspects more of the present invention provide quick for infected by microbes and temporary biophylaxis strengthens or amplification.The enhancing of experimenter's immunity has alleviated infected by microbes.Alleviating can be to realize by the inhibition to infection or growth of microorganism or survival, treatment or prevention.Aspects more of the present invention have strengthened the defence of experimenter's lung and respiratory tract.

In some aspects, consider to treat, suppress or alleviate the method for infected by microbes in suffering from infected by microbes or thering is the individuality of the risk that this infection occurs, described method comprises the StIR compositions of using effective dose, one or more parts that described compositions comprises one or more congenital receptors (innate receptor).Identified many congenital receptors, it includes but not limited to that Toll sample receptor (TLR), C type agglutinin receptor (CLR) and nucleotide are in conjunction with oligomerization domain sample receptor (Nod sample receptor or NLR).TLR is the class protein playing a crucial role in innate immune system.They are non-catalytic receptors of single cross-film, and it identifies conservative molecule in the structure from microorganism.Once these microorganisms are present on skin or intestinal, lung and urogenital tract mucosa or are present in wherein, they identified by TLR, and immune cell activated is replied.What is interesting is, in the time using separately, many these TLR agonist are not induced significant StIR.Conventionally the individuality that, use methods described herein are treated or experimenter have contacted pathogenic microbes or have had the risk of this contact.

Some embodiment relates to the compositions that can use to respiratory tract, and uses that the method for these compositionss, described compositions comprise 1,2,3,4 kind or more kinds of TLR agonist.Described TLR agonist is selected from TLR2/1, TLR2/6, TLR3, TLR4, TLR5, TLR9 or TLR7 agonist.In some aspects, described TLR agonist is selected from TLR9 and TLR2/6 agonist.On the other hand, described TLR agonist is selected from TLR5 agonist.Aspect another, TLR5 agonist can be used in combination with TLR2/6, TLR4, TLR9 or TLR7 agonist.In some aspects, TLR9 agonist can be used in combination with TLR2/6, TLR4, TLR5 or TLR7.On the other hand, TLR2/6 agonist can be used in combination with TLR4, TLR5, TLR9 or TLR7 agonist.In some aspects, TLR4 agonist can be used in combination with TLR2/6, TLR5, TLR9 or TLR7 agonist.On the other hand, TLR7 agonist can be used in combination with TLR2/6, TLR4, TLR5 or TLR9 agonist.Aspect another, any these combination of two can comprise the third or the 4th kind or the 5th kind of TLR agonist that are selected from TLR2/6, TLR4, TLR5, TLR9 or TLR7 agonist.

Some embodiment relates to the method for the treatment of, suppressing or alleviate infected by microbes, and it comprises to suffering from infected by microbes or having the individuality that occurs or obtain the risk of infected by microbes to use TLR9 agonist and the TLR2/6 agonist of effective dose.In some aspects, described TLR2/6 agonist is PAM2CSK4.On the other hand, described TLR9 agonist is C type oligodeoxynucleotide (oligodeoxynucleotide, ODN).C type ODN can include but not limited to ODN2395 or ODNM362 or ODN10101 or another C type ODN or its analog.In some aspects, experimenter has contacted pathogenic microbes or has had the risk that contacts pathogenic microbes.Described microorganism can be virus, antibacterial or fungus.

Aspect other, use described TLR9 agonist and TLR2/6 agonist with spray agent.Use described TLR9 agonist and/or TLR2/6 agonist with approximately 0.1,1,5,10,50 μ g or mg/kg to the amount of approximately 5,10,50,100 μ g or mg/kg whose body weight (comprising all numerical value and scope therebetween).

Some embodiment relates to pharmaceutically acceptable compositions, and it comprises TLR9 agonist and TLR2/6 agonist, antiinflammatory and one or more pharmaceutical excipients, and wherein said compositions is aseptic and does not basically contain pathogenic microbes.In some aspects, described TLR2/6 agonist is PAM2CSK4.On the other hand, described TLR9 agonist is C type oligodeoxynucleotide (ODN).C type ODN can include but not limited to ODN2395 or ODNM362 or ODN10101.

In some aspects, described StIR compositions comprises flagellin polypeptide or its section or derivant, 10,11,12,13,14,15,16,17,18,19,20,21 or 22 continuous amino acids that described flagellin polypeptide comprises the peptide QRLSTGSRINSAKDDAAGLQIA (SEQ ID NO:2) that is called TLR5 agonist.Polypeptide of the present invention also can comprise the aminoacid sequence with SEQ ID NO:2 with at least 70,80 or 90% (comprising all values and scope therebetween) homogeneity.Aspect other, flagellin is flagellin polypeptide or peptide that synthesize and/or purification or that separate.Term " purification " or " separation " represent this component be before from other protein or synthetic reaction thing or by-product isolated or purified, and the purity of described component before being formulated in compositions is at least about 95%.In certain embodiments, the purity of the component of described purification or separation is about or is at least about 80,95,96,97,98,99,99.1,99.2,99.3,99.4,99.5% or higher, or any scope that wherein can derive.Then can be by mixed to the component of this purification and other components, to form compositions as herein described.

5,10,15,20,21,22,23,24,25,30,35,40,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350 or 400 continuous amino acids that restructuring flagellin or its fragment or section comprise SEQ ID NO:2 or other flagellin polypeptide, comprise all values and scope therebetween.These fragments or section and SEQ ID NO:2 or other flagellin polypeptide have at least, at the most or approximately 70,75,80,85,90,95,96,97,98,99 or 100% homogeneity.In some aspects, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 75% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 80% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 85% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 90% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 95% homogeneity.The derivant of flagellin or its section or variant comprise insertion, disappearance and the point mutation to SEQ ID NO:2.A kind of specific insertion mutation is fusion rotein, and it comprises flagellin at c-terminus or aminoterminal is the aminoacid sequence of external source.Many flagellins are known in the art, include but not limited to the flagellin BAB58984 (gi|14278896) of following accession number; YP_001330159 (gi|150402865); YP_001323483 (gi|150399716); CAA28975 (gi|1333716); CAA02137 (gi|1567895); CAA67105 (gi|1580779); AAR10473 (gi|38049688); CAR58992 (gi|197093531); YP_001217666 (gi|147675484); CAL12564 (gi|122089712); BAD14977 (gi|46093563); Or CAD05707 (gi|16503200), all to quote mode, by it, the content whole when the application's priority date is incorporated to herein.

Embodiments more of the present invention can be used by respiratory tract.Method of the present invention comprises by suction or other medications uses compositions to upper respiratory tract and/or lower respiratory tract.In some aspects, use by suction.In some aspects, described StIR compositions is used in spray or aerosol.On the other hand, described compositions be aerosolization or atomization, or can be inhaled into or splash into the form in experimenter.Described compositions can be by sucking or air-breathing using.Can approximately 0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,35,40,45,50,55,60,65,70 μ g or mg/kg use described StIR compositions to the amount of approximately 55,60,65,70,75,80,85,90,95,100,125,150,200 μ g or mg/kg whose body weight, comprise TLR agonist independent or coagulation.Aspect other, can use to experimenter StIR or every kind of TLR agonist or the whole TLR agonist of approximately 0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,200 μ g or mg.Described experimenter can be the risk that has virus, antibacterial or the fungus of contact suction, or has contacted virus, antibacterial or the fungus of suction.Another embodiment comprises such method, wherein before the risk of contact or doubtful contact organism or contact organism improves; Afterwards; In process; Before and afterwards; Before with process in; After process neutralization; Before, afterwards with process in applying said compositions.Described experimenter can contact biological weapons or conditionality pathogen.In some particular aspects, experimenter is immunocompromised host, as cancer patient or AIDS patient.

In yet another embodiment, the present invention relates to pharmaceutically acceptable compositions, it comprises one or more TLR agonist; Antiinflammatory; Antimicrobial; And/or one or more pharmaceutical excipients.Conventionally, such composition is aseptic, and does not basically contain pathogenic microbes.

Pathogenicity that treat in some aspects, or that protect for it or potential pathogenic microbes are virus, antibacterial and/or fungus.In some aspects, microorganism is virus.Virus can be from Adenoviridae (Adenoviridae), coronaviridae (Coronaviridae), filamentous virus section (Filoviridae), flaviviridae (Flaviviridae), Hepadnaviridae (Hepadnaviridae), herpetoviridae (Herpesviridae), orthomyxoviridae family (Orthomyxoviridae), Paramyxoviridae (Paramyxovirinae), Pneumovirinae (Pneumovirinae), Picornaviridae (Picornaviridae), Poxviridae (Poxyiridae), the virus of Retroviridae (Retroviridae) or Togaviridae (Togaviridae), and/or parainfluenza virus (Parainfluenza), influenza virus (Influenza), H5N1, Marburg virus (Marburg), Ebola virus (Ebola), Severe acute respiratory syndrome coronavirus, yellow fever virus, human respiratory syncytial precursor virus, Hantaan virus (Hantavirus) or vaccinia virus.

Aspect another, pathogenicity or the potential pathogenic microorganism for the treatment of or protect for it are antibacterials.Antibacterial can be intracellular bacteria, gram positive bacteria or gram negative bacteria.On the other hand, described antibacterial includes but not limited to staphylococcus (Staphylococcus), bacillus cereus (Bacillus), Frances Salmonella (Francisella) or yersinia (Yersinia).Aspect another, described antibacterial is that Bacillus anthracis (Bacillus anthracis), Yersinia pestis (Yersinia pestis), soil are drawn hot Frances Salmonella (Francisella tularensis), Pseudomonas aeruginosa (Pseudomonas aerugenosa) or staphylococcus aureus (Staphylococcus aureas).In certain embodiments, antibacterial is Bacillus anthracis and/or staphylococcus aureus.Aspect another, described antibacterial is drug resistance bacterium, as multi-drug resistance staphylococcus aureus (multiple drug resistant Staphylococcus aureas, MRSA).Representational medical science dependency gram negative bacteria comprises hemophilus influenza (Hemophilus influenzae), Klebsiella pneumonia (Klebsiella pneumoniae), legionella pneumophila (Legionella pneumophila), Pseudomonas aeruginosa, escherichia coli (Escherichia coli), proteus mirabilis (Proteus mirabilis), enterobacter cloacae (Enterobacter cloacae), serratia marcesens (Serratia marcescens), helicobacter pylori (Helicobacter pylori), Salmonella enteritidis (Salmonella enteritidis) and Salmonella typhi (Salmonella typhi).Representative gram positive bacteria includes but not limited to bacillus cereus, Listerella (Listeria), staphylococcus, streptococcus (Streptococcus), enterococcus (Enterococcus), actinomycetes (Actinobacteria) and clostridium (Clostridium), lack cell wall and can not Gram-stained mycoplasma (Mycoplasma), comprises those antibacterials from these forms.

Aspect another; pathogenicity that treat or that protect for it or potential pathogenic microbes are funguses, as the member of aspergillosis (Aspergillus), candida mycoderma (Candida), cryptococcus (Crytpococus), histoplasma capsulatum (Histoplasma), coccidioides immitis (Coccidioides), blastomyces (Blastomyces), lung sac worm (Pneumocystis) or zygomycete (Zygomyces) class.In another other embodiments, fungus includes but not limited to Aspergillus fumigatus (Aspergillus fumigatus), Candida albicans (Candida albicans), Cryptococcus histolyticus (Cryptococcus neoformans), histoplasma capsulatum (Histoplasma capsulatum), Blastomyces coccidioides (Coccidioides immitis) or Pneumocystis carinii (Pneumocystis carinii).Zygomycetes comprises the mould order of frog excrement (Basidiobolales) (Basidiobolaceae (Basidiobolaceae)), the mould order of two pearls (Dimargaritales) (Dimargaritaceae (Dimargaritaceae)), the mould order of capsula interna (Endogonales) (Endogone (Endogonacea)), Entomophthorales (Entomophthorales) (the mould section of crescent (Ancylistaceae), Jue Mei section (Completoriaceae), Entomophthoraceae (Entomophthoraceae), Ding Liemei section (Meristacraceae), Xin Jiemei section (Neozygitaceae)), comb mould order (Kickxellales) (Shu Mei section (Kickxellaceae)), Mortierella order (Mortierellales) (Mortierellaceae (Mortierellaceae)), Mucorales (Mucorales) and insect-catching Zoopagales (Zoopagales).Aspergillosis class includes but not limited to light blue grey aspergillosis (Aspergillus caesiellus), aspergillus candidus (A.candidus), Aspergillus carneus (A.carneus), rod aspergillosis (A.clavatus), Aspergillus deflectus (A.deflectus), Aspergillus flavus (A.flavus), Aspergillus fumigatus (A.fumigatus), Aspergillus glaucus (A.glaucus), aspergillus nidulans (A.nidulans), aspergillus niger (A.niger), Aspergillus ochraceus (A.ochraceus), aspergillus oryzae (A.oryzae), aspergillus parasiticus (A.parasiticus), broom shape (A.penicilloides), Aspergillus restrictus (A.restrictus), Aspergillus sojae (A.sojae), aspergillus sydowi (A.sydowi), A.tamari, aspergillus terreus (A.terreus), aspergillus ustus (A.ustus), aspergillus versicolor (A.versicolor) etc.Candida mycoderma family includes but not limited to Candida albicans, candida dubliniensis (C.dubliniensis), Candida glabrata (C.glabrata), candida guilliermondii (C.guilliermondii), candida kefyr (C.kefyr), monilia krusei (C.krusei), Candida lusitaniae (C.lusitaniae), C.milleri, C.oleophila, Candida parapsilosis (C.parapsilosis), candida tropicalis (C.tropicalis), Candida utilis (C.utilis) etc.

In some aspects, pathogen is intracellular bacteria, gram positive bacteria or gram negative bacteria.In certain embodiments, described antibacterial is streptococcus, staphylococcus, bacillus cereus, Frances Salmonella or yersinia.In aspect other, described antibacterial is that Bacillus anthracis, Yersinia pestis, soil are drawn hot Frances Salmonella, streptococcus pneumoniae (Streptococcus pnemoniae), staphylococcus aureus, Pseudomonas aeruginosa and/or Burkholderia cepacia (Burkholderia cepacia).

During for claims and/or description, term " alleviates ", any modification of " inhibition ", " reduction " or " preventing " or these terms comprises any measurable reduction or suppresses completely, with the result that obtains wanting, for example microbial load after contact or growth reduce.

In claims and/or description, " comprise " while being combined with term, the noun that countless measure word are modified can refer to one/kind, but also consistent with " one/kind or multiple/kind ", " at least one/kind " and " one 's/kind or more than one/kind " the meaning.

Any embodiment of discussing herein can be implemented by any method of the present invention or compositions, and vice versa.In addition the method that, compositions of the present invention and medicine box can be used in the present invention.

In this application, term " about " is used for representing numerical value, comprises the standard deviation of the error of device for measuring this value or method.

The use of the term "or" in claim is used for representing "and/or", unless show clearly only to refer to that alternative or alternative are mutual exclusions, but present disclosure support only refers to the definition of alternative and "and/or".Comprise "and/or", "or" or " with " some enumerate, one or more listed members can foreclose clearly from described enumerating.

As used in the specification and claims, word " comprises ", " having ", " comprising " or " containing " comprise formula or open, and do not get rid of other NM key element or method steps.

Other targets of the present invention, Characteristics and advantages can become more obvious from the following detailed description.But should understand, although detailed description and specific embodiment have shown particular of the present invention, but they are only for illustration purpose, because based on this detailed description, the multiple change in spirit and scope of the invention and modification are clearly for a person skilled in the art.

Accompanying drawing is described

The following drawings forms the part of this description, and is included for further illustrating some aspect of the present invention.By reference to one or more in these accompanying drawings and in conjunction with the detailed description to specific embodiments presenting herein, the present invention may be better understood.

Fig. 1. natural endotoxin (TLR4 agonist) is induced certain StIR.In the estimation LPS concentration (" endotoxin 1x ") with in NTHI lysate (" NTHi sup "), NTHi lysate, think that LPS in lysate 10 times (" endotoxin 10x ") processes or do not process after 24 hours, with streptococcus pneumoniae (5 × 10 ¹⁰cFU/ml) attack wild type Swiss-Webster mice (10/group).

Fig. 2. synthetic six acyl group lipid As (TLR4 agonist) are not induced StIR.Attacking with Pseudomonas aeruginosa first 24 hours, with synthetic lipid A suspension or PBS processing wild type Swiss-Webster mice (8/group).

Fig. 3. show and carried out first 24 hours of infectivity attack with Pseudomonas aeruginosa, with the imiquimod (TLR7 agonist) of high dose or low dosage or the representativeness of PBS treatment S wiss-Webster mice (8/group) experiment.

Fig. 4. while only having TLR9 to stimulate, induce minimum protection.First 24 hours of the pseudomonas aeruginosa infection sucking in use, with PBS or ODN2395 processing wild type Swiss-Webster mice (8/group).

Fig. 5. utilize the high dose processing that TLR2/6 agonist carries out to induce StIR.Using first 24 hours of pseudomonas aeruginosa infection, with Pam2CSK4 or the PBS processing wild type Swiss-Webster mice of high dose or low dosage.

The combination of Fig. 6 .TLR agonist is than the stronger StIR of any independent agonist induction.With ODN2395 (20 μ g/ml, 8 mices), Pam2CSK4 (20 μ g/ml, 8 mices), two kinds of agonist (10 mices) or PBS (10 mices) processing wild type Swiss-Webster mice.

Fig. 7. synthetic flagellin fragment (TLR5 agonist) induction StIR.With first 24 hours of pseudomonas aeruginosa infection, by the high conservative section of 22 aminoacid of flagellin or only have PBS aerosolization in wild type Swiss-Webster.

Fig. 8. influenza A virus/HK lung consolidated material 11-29-05 aerosol infects the impact on body weight: an aerosol processing in 30 minutes; Influenza virus dosage :～100 times of TCID ₅₀/ mice.When body weight starts, decline with the development of infecting, reflected the order of severity of disease, then in recovery process, rise.

Fig. 9. influenza A virus/HK lung consolidated material 11-29-05 aerosol infects the impact on survival: an aerosol processing in 30 minutes; Influenza virus dosage :～100 times of TCID ₅₀/ mice.

Figure 10. an aerosol pretreatment in 30 minutes having shown ODN/PAM2/PolyIC is on infecting the impact of mouse survival of influenza A virus/HK aerosol; Virus dosage～130 times TCID ₅₀/ mice.

Figure 11. influenza A virus/HK lung consolidated material 11-29-05 aerosol infects the impact on body weight: an aerosol processing in 30 minutes; Influenza virus dosage :～100 times of TCID ₅₀/ mice.When body weight starts, decline with the development of infecting, reflected the order of severity of disease, then in recovery process, rise.

What the pneumonia resistance of Figure 12 A and the induction of 12B. antibacterial lysate needed is MyD88 (rather than TRIF) signal transduction.Figure 12 A., sucked and attacks Myd88 with Pseudomonas aeruginosa with atomization lysate pretreatment that can not typing hemophilus influenza (NTHi) or do not carry out in pretreated situation at 24 hours before ^-/-and wild-type mice.A left side, survival (N=10 mice/group, ^*p < 0.0001).The right side, the lung bacterial load occurring immediately after infection (right side, N=3 mice/group, ^*p < 0.004, with respect to wild type contrast).Figure 12 B. uses in antibacterial lysate pretreatment or not pretreated situation, Trif ^-/-the Pseudomonas aeruginosa of mice is attacked.A left side, survival (N=10 mice/group, ^*p < 0.0001).The right side, the lung bacterial load occurring immediately after infection (right side, N=3 mice/group, ^*p < 0.0001).

Figure 13. the pathogen of inducing is killed and wounded in interleukin 1 receptor deficient mice not impaired.Attacking with Pseudomonas aeruginosa first 24 hours, using the PBS of aerosolization or can not process I by typing hemophilus influenza (NTHi); Lr ^-/-and wild-type mice.The bacterial load of the lung homogenate that shown is occurs after infecting immediately.(N=3 mice/group, ^*p=0.001 is with respect to wild type+PBS, ^*p=0.01 is with respect to Illr ^-/-,

with respect to wild type+PBS,

with respect to wild type+NTHi).

Figure 14. the numeration of leukocyte after processing with single synthetic TLR part in bronchoalveolar lavage fluid.Process after 24 hours with one of PBS or following TLR part, mice is carried out to BAL:Pam3CSK4 (TLR2/1 agonist, 1 μ g/ml, 3 μ g/ml, 10 μ g/ml), Pam2CSK4 (TLR2/6 agonist, 1 μ g/ml, 3 μ g/ml, 10 μ g/ml), Poly (I:C) (TLR3 agonist, 1 μ g/ml, 10 μ g/ml, 100 μ g/ml), synthetic lipid A (MPLA, TLR4 agonist, 1 μ g/ml, 10 μ g/ml, 100 μ g/ml), Flg22 (synthetic 22-mer flagellin, TLR5 agonist, 10 μ g/ml, 100 μ g/ml, 1000 μ g/ml), imiquimod (TLR7 and TLR8, 100 μ g/ml, 300 μ g/ml, 1000 μ g/ml) or ODN2395 (TLR9 agonist, 2 μ g/ml, 20 μ g/ml).Shown is neutrophil cell (black bar) and the macrophage (grey bar) in BAL liquid.

Figure 15 A-15G. utilizes single synthetic TLR part to carry out aerosolization processing and does not induce high-caliber pneumonia resistance.With PBS or following synthetic TLR part processing (8ml spraying, in 20 minutes, use) 24 hours afterwards, utilize Pseudomonas aeruginosa to attack wild-type mice: Figure 15 A.TLR2/1 agonist Pam3CSK4100 μ g/ml, Figure 15 B.TLR2/6 agonist Pam2CSK410 μ g/ml, Figure 15 C.TLR3 agonist poly (I:C) 100 μ g/ml, Figure 15 D.TLR4 agonist MPLA100 μ g/ml, Figure 15 E.TLR5 agonist Flg22100 μ g/ml, Figure 15 F.TLR7 and TLR8 agonist imiquimod 1mg/ml, or Figure 15 G.TLR9 agonist ODN239520 μ g/ml.Survival curve be process and the representative example of at least three group different experiments of untreated mice (N=8 mice/group, ^*p=0.5, ^*p=1.0,

).

The resistance of Figure 16 A-16C.TLR2/6 and TLR9 agonist cooperation induction directed toward bacteria property pneumonia.Figure 16 A. left side, with after the combined treatment of PBS, Pam2CSK410 μ g/ml, ODN239520 μ g/ml, combination or doubling dosage 24 hours, the survival of the mice of attacking with Pseudomonas aeruginosa (N=6 mice/group,

than PBS).The right side, the bacterial load (N=3 mice/group, #p=0.045 is than PBS, ##p=0.030 is than PBS) of the lung homogenate producing immediately after pseudomonas aeruginosa infection.Figure 16 B. left side, with after PBS, Pam2CSK410 μ g/ml, ODN239520 μ g/ml, combination or doubling dosage combined treatment 24 hours, the survival of the mice of attacking with streptococcus pneumoniae (N=10 mice/group,

process than PBS).The right side, streptococcus pneumoniae infection 2 × 10 ¹⁰after immediately produce lung homogenate bacterial load (N=3 mice/group,

).After the combined treatment of Figure 16 C.PBS, Pam2CSK410 μ g/ml, ODN239520 μ g/ml or Pam2CSK4 and ODN2395 4 or 24 hours, from the BAL cell counting of mice (N=3 mice/group, ^*p=0.016 is than PBS, ^*p < 0.0001 is than PBS,

than only there being Pam2).

Figure 17 A-17F. is not that all TLR agonist combinations all provide the remarkable protection for pneumonia.Utilize after PBS or following TLR agonist combined treatment 24 hours, attack wild-type mice with Pseudomonas aeruginosa: Figure 17 A.Pam2CSK4 and poly (I:C), Figure 17 B.Pam2CSK4 and Flg22, Figure 17 C.Pam2CSK4 and imiquimod, Figure 17 D.ODN2395 and poly (I:C), Figure 17 E.ODN239 and Flg22, Figure 17 F.ODN2395 and Pam3CSK4.Survival curve be at least three different experiments representative example (N=8 mice/group, ^*p=0.20, ^*p=0.08,

).

Figure 18 A-18B.TLR2 is enough to promote protectiveness Pam2CSK4 and ODN2395 synergism, but is not that reactance power is necessary.Figure 18 A. left side, with or process latter 24 hours without ODN2395 and Pam2CSK4, with Pseudomonas aeruginosa attack Tlr2 ^-/-with the survival of wild-type mice (N=8 mice/group, ^*p < 0.0002).The right side, with after pseudomonas aeruginosa infection immediately occur lung homogenate bacterial load (N=4 mice/group, ^*p < 0.0001 is than wild type+PBS,

than Tlr2 ^-/-+ PBS).Figure 18 B. left side, with or atomization lysate that need not typing hemophilus influenza (NTHi) process latter 24 hours, attack TLR2 with Pseudomonas aeruginosa ^-/-with the survival of wild-type mice (N=10 mice/group, ^*p < 0.0002).The right side, with after pseudomonas aeruginosa infection immediately occur lung homogenate bacterial load (N=3 mice/group, than wild type+PBS, #p=0.002 is than Tlr2 ^-/-+ PBS).

CpG ODN and Pam2CSK4 synergism that Figure 19 A-19B.C type (rather than category-A and category-B) is combined with TLR9, to induce the resistance to bacterial pneumonia.Figure 19 A. Pseudomonas aeruginosa attack before 24 hours, the survival of the wild-type mice of processing with Pam2CSK4 and ODN2395 or Pam2CSK4 and out of order contrast ODN (N=10 mice/group, ^*p < 0.0001).FIG.19B. process latter 24 hours with PBS or Pam2CSK4 combination category-A CpG ODN (ODN1585 or ODN2216), category-B CpG ODN (ODN2006-G5) or C class CpGODN (M362 or ODN2395), survival (N=10 the mice/group of the wild-type mice of attacking with Pseudomonas aeruginosa ^*p=0.01 is than PBS, ^*p=0.0001 is than PBS;

than Pam2+ODN2395).

Figure 20 A-20D.TLR2/6 and TLR9 agonist in vitro co-induction mice and the epithelial antibacterial of human respiratory kill and wound.Figure 20 A. uses Pam2CSK4 (10 μ g/ml) and/or ODN2395 (20 μ g/ml) to process MLE-15 cell 4 hours before infecting with Bacillus anthracis (1000 spore).Shown is infect latter 4 hours antibacterial CFU ( ^*p=0.05 is than PBS, ^*p=0.016 is than PBS, and #p > 0.05 is than arbitrary single agonist).Figure 20 B. processes MLE culture medium (acellular) with ODN2395 and Pam2CSK4, after 4 hours, infects and cultivates with Bacillus anthracis (1000 spore)

figure 20 before Pseudomonas aeruginosa (2700CFU) infection, uses ODN2395 and Pam2CSK4 to process A549 cell 4 hours for C..Shown is infect latter 4 hours antibacterial CFU ( ^*p=0.01 is than PBS, ^*p=0.003 is than PBS, ^{* *}p=0.001 is than PBS, and #p=> 0.05 is than arbitrary single agonist).Figure 20 D. processes MLE culture medium (acellular) with ODN2395 and Pam2CSK4, after 4 hours, infects and cultivates with Pseudomonas aeruginosa (4000CFU)

Figure 21. the survival of the Swiss-Webster mice of also attacking with 5 times of LD50 Bacillus anthracis Ames spore (MD-10-013) intranasal with multiple synthetic TLR agonist immunity.As shown, attacking by anthrax first 24 hours, by the TLR agonist pretreatment of mice of aerosolization.ALIIS=NTHi antibacterial lysate, 2395=ODN2395,10101=ODN10101, the ODN of M362=ODN-M362.1 ×=40 μ g/ml and the Pam2 of 20 μ g/ml.

Figure 22. utilize ODN/Pam2 or NTHi lysate to carry out the impact of the mouse survival of aerosol pretreatment on influenza A virus/HK infection.An aerosol processing in 30 minutes; Influenza virus dosage :～100 times of TCID ₅₀/ mice.

Detailed Description Of The Invention

Immune system is the biological system of avoiding specialized cell and the organ of extraneous biotic influence of protection.In the time that function of immune system is normal, its protection body is avoided infected by microbes, and destruction of cancer cells and foreign body.If immune system weakens, the ability of its protection body also weakens, and makes the pathogen can be at machine tumor growth.

Immune system is often divided into: (a) innate immunity, it comprises provides the directly component of " First Line " defence, to continue to stop pathogen, (b) adaptability (acquired) immunity, the generation that it comprises antibody and be designed for specifically generation or the stimulation of the T cell of targeting special pathogen.Utilize adaptive immunity, health can produce the specific immunity for special pathogen after certain hour.This is replied needs a couple of days to occur, and invalid to preventing from attacking first, but conventionally can prevent any follow-up infection, also helps to remove to continue infection more of a specified duration.

Respond to some inflammatory stimulus, there is rapidly significant structural change in the secretory cell of mice and human respiratory epithelium, is called inflammatoryization raw.Most of structural changes are to increase owing to forming the mucinous generation of the secreted of gel, simultaneously mucin secretion, microorganism kill and wound or Inflammatory Signal Transduction in play function other macromole also raise.Think that this physiological function of replying is the local defense of booster injection to microbial pathogens, but the official testing that this hypothesis has been accepted is limited.Self-contradictory, the mucinous excessive generation and the secretion that form gel are the main causes that in the common inflammatory diseases of respiratory tract (as asthma, cystic fibrosis and chronic obstructive pulmonary disease (COPD)), air-flow stops up.Stimulation does not produce mucinous innate immunity will provide a kind of additive method that alleviates respiratory tract infection by preventing and/or treating experimenter.

The air flue that embodiments more of the present invention comprise with comprising 1,2,3, the compositions of 4 kind or more kinds of TLR agonist (comprising its section or derivant or analog) stimulates experimenter.The experimenter who uses the present composition is endowed for the therapeutic of latent infection biology, preventative or therapeutic and preventative replying.In aspect some are concrete, by compositions atomization and use by respiratory tract.Described compositions is for for example inducing by the innate immunity activating or strengthen lung or otherwise causing protective effect.

Some aspect of the present invention comprises micromolecule and/or TLR agonist, and it is from different microorganisms, or synthetic.Conventionally, described micromolecule and/or TLR agonist do not cause that the mucinous generation of secreted increases.Embodiment of the present invention can be used as for the preventative of biological example weapon, new poisonous microorganism or conditionality microorganism and property (preemptive) therapeutic agent in advance.

I.StIR compositions

A. allos compound and part

The generation that many nonhosts or heterologous molecule can stimulate, enhancing or Promote immunity are replied.These parts comprise multiple congenital receptor stimulating agent and/or microbial components.

1. congenital receptors ligand

Pattern recognition receptors (or is called PRR, pattern recognition receptor) (congenital receptor) be the protein that is used for identifying pathogen associated molecular pattern (or being called PAMP, pathogen-associated molecular pattern) (it coerces relevant to microbial pathogens or cell) of the cellular expression of innate immune system.PAMP (for example includes but not limited to Bacterial Carbon hydrate, lipopolysaccharide or LPS, mannose), nucleic acid (for example, antibacterial or viral DNA or RNA), Peptidoglycan and lipoteichoic acid (from gram positive bacteria), N-formylmethionine, lipoprotein, fungus glucosan etc.

PRR classifies according to its ligand specificity, function, location and/or evolutionary relationship conventionally.Based on function, PRR can be divided into endocytosis PRR or signal transduction PRR.Signal transduction PRR comprises the extended familys of film in conjunction with Toll sample receptor and kytoplasm NOD sample receptor.Endocytosis PRR promotion phagocyte is adhered to, eats and destroy microorganism, but does not transmit intracellular signal.These PRR identification carbohydrates, and comprise macrophage mannose receptor, be present in the glucosan receptor in all phagocyte, and be present in the scavenger receptor (scavenger receptor) of the charged part of identification of all macrophages the removing of mediated apoptosis cell.

Many congenital receptors are identified, the oligomerization domain sample receptor (Nod sample receptor or NLR) that includes but not limited to Toll sample receptor (TLR), C type agglutinin receptor (CLR) and be combined with nucleotide.TLR is the class protein playing a crucial role in innate immune system.They are non-catalytic receptors of single cross-film, and it identifies conservative molecule in the structure from microorganism.Once these microorganisms are present in skin or intestinal mucosa on it or wherein, their be activated TLR of immune cell responses identify.What is interesting is, in the time using separately, many these TLR agonist are not induced significant StIR.Conventionally, use individuality or the experimenter of methods described herein treatment to contact pathogenic microbes, or have the risk of this contact.

A.Toll sample receptor (TLR) agonist

Toll sample receptor (TLR) is the PRR (Ishii etc., 2008) the most fully characterizing.They are transmembrane proteins of high conservative, are made up of ectodomain (ectodomain) (have for the multiple rich leucine of pattern recognition and repeat), transmembrane spanning α-helices and Toll/ interleukin-1 receptor (TIR) domain for intracellular signal transduction.Identified at least 13 kinds of mammal TLR, each equal specific localization is on plasma membrane or endosome film, and each detects unique homologue (Akira etc., 2006 in PAMP; Shi etc., 2006).After PAMP identification, raise generation signal transduction by the TLR specificity of cytosol TIR adaptin combination.With together with one or more in four kinds of other adaptins, TIR adaptin MyD88 is that to start from the signal transduction of most of TLR required.The signal transduction event that does not rely on MyD88 of observing from TLR3 and TLR4 needs TIR adaptin TRIF (also referred to as TICAM-1), and it is with or without the participation (Yamamoto etc., 2003) of TRAM.TLR specificity T IR adaptin signal cascade activated receptor idiosyncratic transcription factor is as NF-κ B, activator protein-1 and interferon regulatory factor (IRF), the expression (Akira etc., 2006 that cause proinflammatory gene and antimicrobial gene; O ' Neill, L.A., and Bowie, 2007; Takeda, K., and Akira, 2004).

TLR agonist is any compound or the material that performance activates the function of TLR (thereby signal transduction event that for example induction mediates by TLR signal transduction pathway).Suitable TLR agonist comprises TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist and TLR9 agonist.

Extensively be recognized that at present, the generation of protective immunity not only depends on antigen contact, also depends on the environment while running into antigen.There are many examples, wherein in inflammatory environment, in host, introduce neoantigen generation immunologic tolerance rather than permanent immunity, contact induction of immunity (Mondino etc., 1996 of antigen under inflammatory reagent (adjuvant) exists; Pulendran etc., 1998; Jenkins etc., 1994; With Keamey etc.).Because this can mean the difference between tolerance and immunity, so paid " adjuvant " of a large amount of effort for finding to exist in infector, it stimulates the molecular pathways that participates in the suitable immunogenicity environment that produces antigen presentation.Be known that at present a large amount of adjuvanticities are due to interaction (Beutler etc., 2004 between the different members of the Toll sample receptor (TLR) of expressing on microorganism and viral product and immunocyte; Kaisho, 2002; Akira etc., 2003; And Takeda and Akira, 2004).TLR gain the name homology (Lernaitre etc., 1996 of the molecule (be called Toll, it brings into play function and participate in antimicrobic immunity in fruit bat grows) in itself and fruit bat; With Hashimoto etc., 1988).

Early stage work demonstration, the mammal congener of Toll and Toll approach molecule are for the ability of innate immune system cell response microorganism attack and microorganism by-product most important (Medzhitov etc., 1997; Medzhitov etc., 1998; Medzhitov etc., 2000; With Janeway etc., 2002).Since LPS is accredited as to TLR4 agonist (Poltorak etc., 1998), many other TLR agonist are described, as trigalloyl Quito type HPV polypeptide (TLRI), Peptidoglycan, lipoteichoic acid and Pam ₃cys (TLR2), dsRNA (TLM), flagellin (TLR5), diacyl polymorphic type HPV polypeptide, as Malp-2 (TLR6), imidazole quinoline and single stranded RNA (TLR7,8), DNA of bacteria, non-methylated CpG DNA sequence, or even human gene group DNA's antibody complex (TLR9) (Takeuchi etc., 2001; Edwards etc., 2002; Hayashi etc., 2003; Nagase etc., 2003).

As used herein, term " agonist " refer to can with the compound of receptor (for example, TLR) combination results cytoactive.Agonist can be the part of direct bind receptor.Or, agonist can combine indirectly by following and receptor, for example, (a) forms complex with another molecule of direct bind receptor, or (b) otherwise cause the modification of other compounds, make the described direct bind receptor of other compounds.Agonist can be called the agonist (for example, the TLR7/8 agonist agonist of TLR7 and TLR8 one by one) of the agonist (for example, TLR7 agonist) of specific T LR or specific T LR combination.

The term " CpG-ODN ", " CpG nucleic acid ", " the CpG polynucleotide " and " CpG ODN " that are used interchangeably herein refer to such polynucleotide, it comprises at least one 5 '-CG-3 ' part, and comprises in many embodiments unmethylated 5 '-CG-3 ' part.Generally speaking, CpG nucleic acid is strand or double-stranded DNA or the RNA polynucleotide with at least 6 nucleotide bases, and it can comprise the nucleotide of modification or the nucleotide sequences of modification, or consisting of.In some embodiments, the 5 '-CG-3 ' of CpG nucleic acid part is a part for palindrome nucleotide sequence.In some embodiments, the 5 '-CG-3 ' of CpG nucleic acid part is a part for non-palindrome nucleotide sequence.

Suitable TLR agonist comprises the natural TLR agonist of separation, and synthetic TLR agonist.The TLR agonist separating from natural TLR agonist source is generally purification, and the purity of the TLR agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR agonist is prepared by standard method, and general purity is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

Suitable TLR agonist comprises the TLR agonist being not adhered on any other compound.Suitable TLR agonist comprises the TLR agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR agonist is directly attached on another compound.In other embodiments, TLR agonist is attached on another compound by joint.The accompanying compound of TLR agonist comprises carrier, support, insoluble holder, microgranule, microsphere etc.Carrier comprises therapeutical peptide; The more polypeptide of high-dissolvability is provided; Improve the polypeptide of such as, half-life in physiological medium (serum or other body fluid) of TLR agonist etc.In some embodiments, TLR agonist will directly or be conjugated on another TLR agonist by joint.

In some embodiments, TLR agonist is the prodrug forms of TLR agonist.Prodrug is by forming with the covalently bound prodrug moiety of active therapeutic agent.Prodrug can change into medicine (active therapeutic agent) by some chemistry or the enzymatically modifying of its structure in vivo.The example of prodrug moiety is known in the art, and be found in below with reference in document: Biological Approaches to the Controlled Delivery of Drugs, R.L.J uliano, New York Academy of Sciences, (1988); Hydrolysis in Drug and Prodrug Metabolism:Chemistry, Biochemistry, and Enzymology, Bernard Testa, Vch Verlagsgesellschaft Mbh, (2003); With Prodrugs:Topical and Ocular Drug Delivery, Kenneth Sloan, Marcel Dekker; (1992).The example of prodrug moiety is peptide, for example, TLR part is directed to the peptide on action site, and has two or more free and peptides of the carboxylic acid of coupling not at its aminoterminal.Other exemplary prodrug moieties that cut comprise ester group, ether, acyl group, alkyl, phosphate, sulfonic group, N oxide and tert-butoxycarbonyl.

In some embodiments, TLR agonist is monomer TLR agonist.In other embodiments, TLR agonist is multimerization, and for example TLR agonist is polymeric.In some embodiments, the TLR agonist of multimerization is congenerous, for example, be made up of the TLR agonist of a type.In other embodiments, the TLR agonist of multimerization is exclusive-OR function TLR agonist.

In some embodiments, TLR part is chimeric TLR part (herein also referred to as " exclusive-OR function " TLR part).In some embodiments, chimeric TLR agonist comprises TLR9 agonist part and TLR2 agonist part.It is below the limiting examples of exclusive-OR function TLR agonist.

In some embodiments, chimeric TLR part has following general formula: (X) n-(Y) m, wherein X is TLR1 agonist, TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist and TLR9 agonist, and Y is TLR2 agonist, TLR3 agonist, TLR4 agonist, TLR5 agonist, TLR6 agonist, TLR7 agonist, TLR8 agonist and TLR9 agonist, n and m are 1 independently, 2, 3, 4, 5, 6, 7, 8, 9, 10 or the integer of larger (comprising all values and scope therebetween).In certain embodiments, X or Y are TLR9, and X or Y are TLR2/6.

TLR2 agonist .TLR2 agonist comprises the natural TLR2 agonist of separation; With synthetic TLR2 agonist.The TLR2 agonist separating from natural TLR2 agonist source is generally purification, and the purity of the TLR2 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR2 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR2 agonist comprises the TLR2 agonist being not adhered on any other compound.TLR2 agonist comprises the TLR2 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR2 agonist is directly attached on another compound.In other embodiments, TLR2 agonist is attached on another compound by joint.

TLR2 agonist comprises the lipopeptid of synthetic triacylated and diacyl.The limiting examples of TLR2 part is FSL-1 (from the synthetic fat albumen of mycoplasma salivarium 1), Pam ₃cys (three palmityls-S-glyceryl cysteine) or S-[2,3-bis-(palmityl oxygen base)-(2RS)-propyl group]-N-palmityl-(R)-cysteine, wherein " Pam ₃" be " three palmityls-S-glyceryl ") (Aliprantis etc., 1999).Pam ₃the derivant of Cys is also suitable TLR2 agonist; wherein derivant includes but not limited to S-[2; 3-bis-(palmityl oxygen base)-(2-R, S)-propyl group]-N-palmityl-(R)-Cys-(S)-Ser-(Lys) ₄-hydroxyl trihydrochloride; Pam ₃cys-Ser-Ser-Asn-Ala; PaM ₃cys-Ser-(Lys) ₄; Pam ₃cys-Ala-Gly; Pam ₃cys-Ser-Gly; Pam ₃cys-Ser; PaM ₃cys-OMe; Pam ₃cys-OH; PamCAG, palmityl-Cys ((RS)-2,3-bis-(palmityl oxygen base)-propyl group)-Ala-Gly-OH etc.Another limiting examples of suitable TLR2 agonist is Pam ₂cSK ₄.PaM ₂cSK ₄(two palmityls-S-glyceryl cysteine-serine-(lysine) ₄or Pam ₂cys-Ser-(Lys) ₄) be synthetic diacyl lipopeptid.Synthetic TLR agonist has been described in list of references.For example consult Kellner etc. (1992); Seifer etc. (1990); Lee etc. (2003).

TLR3 agonist .TLR3 agonist comprises the natural TLR3 agonist of separation; With synthetic TLR3 agonist.The TLR3 agonist separating from natural TLR3 agonist source is generally purification, and the purity of the TLR3 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR3 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR3 agonist comprises the TLR3 agonist being not adhered on any other compound.TLR3 agonist comprises the TLR3 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR3 agonist is directly attached on another compound.In other embodiments, TLR3 agonist is attached on another compound by joint.

TLR3 agonist comprises natural double-stranded RNA (dsRNA); Synthetic dsRNA; With (Alexopoulou etc., 2001) such as synthetic dsRNA analog.The exemplary unrestricted example of synthetic ds RNA analog is poly (I:C).

TLR4 agonist. suitable TLR4 agonist comprises the natural TLR4 agonist of separation; With synthetic TLR4 agonist.The TLR4 agonist separating from natural TLR4 agonist source is generally purification, and the purity of the TLR4 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR4 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR4 agonist comprises the TLR4 agonist being not adhered on any other compound.Suitable TLR4 agonist comprises the TLR4 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR4 agonist is directly attached on another compound.In other embodiments, TLR4 agonist is attached on another compound by joint.The accompanying suitable compound of TLR4 agonist comprises carrier, support etc.

TLR4 agonist comprises natural lipopolysaccharide (LPS), for example, from the LPS of multiple gram negative bacteria; The derivant of natural LPS; Synthetic LPS; Antibacterial HSP60 (Hsp60); Mannuronic acid polymer; Yellow fat element (flavolipin); Teichuronic acid; The pneumolysin (pneumolysin) of streptococcus pneumoniae (S.pneumoniae); Bacterial pilli (fimbriae), respiratory syncytial virus coat protein etc.TLR4 agonist also comprises synthetic LA (MPLA, Invivogen) and phosphorylation six acyl group disaccharide (PHAD, Avanti Polar Lipids), and other synthetic TLR4 agonist.

TLR5 agonist. suitable TLR5 agonist comprises the natural TLR5 agonist of separation; With synthetic TLR5 agonist.The TLR5 agonist separating from natural TLR5 agonist source is generally purification, and the purity of the TLR4 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR5 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR5 agonist comprises the TLR5 agonist being not adhered on any other compound.Suitable TLR5 agonist comprises the TLR5 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR5 agonist is directly attached on another compound.In other embodiments, TLR5 agonist is attached on another compound by joint.The suitable compound that TLR5 agonist adheres to comprises carrier, support etc.

TLR5 agonist comprises 22 aminoacid sections and total length flagellin and other sections thereof of the high conservative of flagellin.

TLR7 agonist. suitable TLR7 agonist comprises the natural TLR7 agonist of separation; With synthetic TLR7 agonist.The TLR7 agonist separating from natural TLR7 agonist source is generally purification, and the purity of the TLR7 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR7 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR7 agonist comprises the TLR7 agonist being not adhered on any other compound.Suitable TLR7 agonist comprises the TLR7 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR7 agonist is directly attached on another compound.In other embodiments, TLR7 agonist is attached on another compound by joint.

TLR7 part comprises imidazole quinoline compound; Guanosine analogue; Pyrimidine compound, as bropirimine and bropirimine analog etc.Rise the imidazole quinoline compound of TLR7 ligand function include but not limited to imiquimod (also referred to as Aldara, R-837, S-26308) and R-848 (also referred to as resiquimod, S-28463; There is following chemical constitution: 4-amino-2-ethoxyl methyl-α, alpha-alpha-dimethyl-1H-imidazoles [4,5-c] quinoline-1-ethanol).Suitable imidazole quinoline agent comprises imidazole quinoline amine, imidazopyridine amine, 6, the cycloalkyl imidazopyridine amine and 1 that 7-condenses, the imidazole quinoline amine that Bridge 2 connects.At United States Patent (USP) 4,689,338,4,929,624,5,238,944,5,266,575,5,268,376,5,346,905,5,352,784,5,389,640,5,395,937,5,494,916,5,482,936,5,525,612,6, in 039,969 and 6,110,929, these compounds are described.The concrete kind that is applicable to the imidazole quinoline agent in the inventive method comprises R-848 (S-28463); 4-amino-2 ethoxyl methyl-α, alpha-alpha-dimethyl-1H-imidazoles [4,5-c] quinoline-s-i-ethanol; And 1-(2-methyl-propyl)-1H-imidazoles [4,5-c] quinolin-4-amines (R-837 or imiquimod).That be also applicable to using is compound 4-amino-2-(ethoxyl methyl)-α, alpha-alpha-dimethyl-6, and 7,8,9-tetrahydrochysene-1H-imidazoles [4,5-c] quinoline-1-alcohol hydrate (is for example consulted BM-003, Gorden etc. (2005).

Suitable compound comprises PA and five Yuans those compounds that nitrogen heterocyclic ring condenses.This compounds comprises as imidazole quinoline amine, include but not limited to the imidazole quinoline amine replacing, the imidazole quinoline amine of the imidazole quinoline amine that the imidazole quinoline amine that for example amide replaces, the imidazole quinoline amine of sulfonamide substitutions, carbamide replace, the imidazole quinoline amine that aryl ether replaces, heterocyclic ether replacement, the imidazole quinoline amine that amino ethers replaces, the imidazole quinoline amine that sulphonyl amidogen ether replaces, the imidazole quinoline ether that carbamide replaces, the imidazole quinoline amine that thioether replaces, and the imidazole quinoline amine of 6-, 7-, 8-or 9-aryl or heteroaryl replacement; Imidazolidine quinolinamine, includes but not limited to imidazolidine quinolinamine, the imidazolidine quinolinamine of sulfonamide substitutions, the imidazolidine quinolinamine of carbamide replacement, the imidazolidine quinolinamine that aryl ether replaces, the imidazolidine quinolinamine that heterocyclic ether replaces, the imidazolidine quinolinamine that amino ethers replaces, the imidazolidine quinolinamine that sulphonyl amidogen ether replaces, the imidazolidine quinoline ether of carbamide replacement and the imidazolidine quinolinamine that thioether replaces that amide replaces; Imidazopyridine amine includes but not limited to imidazopyridine amine, the imidazopyridine amine of sulfonamide substitutions, the imidazopyridine amine of carbamide replacement, the imidazopyridine amine that aryl ether replaces, the imidazopyridine amine that heterocyclic ether replaces, the imidazopyridine amine that amino ethers replaces, the imidazopyridine amine that sulphonyl amidogen ether replaces, the imidazopyridine ether of carbamide replacement and the imidazopyridine amine that thioether replaces that amide replaces; 1,2-bridge joint imidazole quinoline amine; The cycloalkyl imidazopyridine amine that 6,7-condenses; Imidazo naphthyridines amine; Imidazolidine naphthyridines amine;

azoles quinolinamine; Thiazole quinolinamine;

azoles pyridine amine; Thiazole pyridine amine;

azoles naphthyridines amine; Thiazole naphthyridines amine; With the 1H-imidazoles dimer, quinolinamine, tetrahydroquinoline amine, naphthyridines amine and the Tetrahydronaphthyridderivates amine that condense with pyridine amine.

Compound comprises imidazole quinoline amine, imidazolidine quinolinamine, the imidazopyridine amine, 1 of replacement, the imidazole quinoline amine, 6 of 2-bridge joint, cycloalkyl imidazopyridine amine that 7-condenses, imidazo naphthyridines amine, imidazolidine naphthyridines amine, azoles quinolinamine, thiazole quinolinamine,

azoles pyridine amine, thiazole pyridine amine,

azoles naphthyridines amine and thiazole naphthyridines amine.

As used herein, the imidazole quinoline amine replacing refers to the imidazole quinoline amine of amide replacement, the imidazole quinoline amine of sulfonamide substitutions, the imidazole quinoline amine that carbamide replaces, the imidazole quinoline amine that aryl ether replaces, the imidazole quinoline amine of heterocyclic ether replacement, the imidazole quinoline amine that amino ethers replaces, the imidazole quinoline amine of sulphonyl amidogen ether replacement, the imidazole quinoline ether that carbamide replaces, the imidazole quinoline amine that thioether replaces, or the imidazole quinoline amine of 6-, 7-, 8-or 9-aryl or heteroaryl replacement.

The guanosine analogue of performance TLR7 ligand function comprises some C8-replaces and N7, the dibasic guanosint ribotide of C8-and deoxyribonucleotide, include but not limited to Loxoribine (7-pi-allyl-8-oxygen guanosine), 7-sulfo--8-oxygen guanosine (TOG), 7-denitrogenation guanosine and 7-denitrogenation deoxyguanosine (Lee etc., 2003).In list of references, described bropirimine (PNU-54461) (5-halo-6-phenyl pyrimidine ketone) and bropirimine analog, and it is also applicable to using.Consult as (1999) such as Vroegop.The additional examples that suitable C8 replaces guanosine includes but not limited to, 8-TGR, 8-bromo guanosine, 8-methylguanosine, 8-oxo-7, the guanosint riboside that 8-dihydro guanosine, amino-the 2 '-deoxyguanosine of C8-virtue, C8-propinyl guanosine, C8-and N7-replace, as 7-pi-allyl-8-oxygen guanosine (Loxoribine) and 7-methyl-8-oxygen guanosine, the amino guanosine of 8-, 8-oxo-dG and 8-hydroxyl guanosine.

In some embodiments, the guanine TLR7 part of replacement is monomer.In other embodiments, the guanine TLR7 part of replacement is poly.Therefore, in some embodiments, TLR7 part has general formula: (B) q, and wherein B is the guanine TLR7 part replacing, q is 1,2,3,4,5,6,7,8,9 or 10.Each TLR7 part monomer in poly TLR7 part directly or be covalently or non-covalently connected with another part by joint.Suitable TLR7 agonist comprises the TLR7 part that discloses description in 2004/0162309 as United States Patent (USP).

In some embodiments, TLR7 agonist is selectivity TLR7 agonist, and for example described agonist regulates cytoactive by TLR7, but does not regulate cytoactive by TLR8.TLR7 selective agonist is included in United States Patent (USP) and discloses those that show in 2004/0171086.This type of TLR7 selective agonist compound includes but not limited to, N ¹-{ 4-[4-amino-2-(2-methoxy ethyl)-6,7,8,9-tetrahydrochysene-1H-imidazoles [4,5-c] quinoline-1-yl] butyl } the fluoro-1-benzsulfamide of-4-, N ¹-[4-(4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4, 5-c] quinoline-1-yl) butyl] the fluoro-1-benzsulfamide of-4-, N-[4-(4-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl) butyl] Methanesulfomide, N-{3-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4, 5-c] quinoline-1-yl]-2, 2-dimethylpropyl } Benzoylamide, N-(2-{2-[4-amino-2-(2-methoxyethyl)-1H-imidazoles [4, 5-c] quinoline-1-yl] ethyoxyl } ethyl)-N-methyl Methanesulfomide, N-(2-{2-[4-amino-2-(2-methoxyethyl)-6, 7, 8, 9-tetrahydrochysene-1H-imidazoles [4, 5-c] quinoline-1-yl] ethyoxyl } ethyl) Benzoylamide, N-[4-(4-amino-2-methyl-1H-imidazole radicals [4, 5-c] quinolyl-1-yl) butyl] ring penta Methanamide, 1-[4-(1, 1-dioxy isothiazolidine-2-yl) butyl]-2-(2-methoxy ethyl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 2-methyl isophthalic acid-[5-methyl sulphonyl] amyl group-6, 7, 8, 9-tetrahydrochysene-1H-imidazoles [4, 5-c] quinolin-4-amines, N-{2-[4-amino-2-(ethoxyl methyl)-6, 7-dimethyl-1H-imidazoles [4, 5-c] pyridine-1-yl]-1, 1-dimethyl ethyl }-N-cyclohexyl urea, N-[2-(4-amino-2-ethyl-1H-imidazoles [4, 5-c] quinoline-1-yl)-1, 1-dimethyl ethyl] Benzoylamide, N-[3-(4-amino-2-butyl-1H-imidazoles [4, 5-c] quinoline-1-yl)-2, 2-dimethyl propyl] Methanesulfomide, 1-[6-(mesyl) hexyl]-6, 7-dimethyl-2-propyl group-1H-imidazoles [4, 5-c] pyridine-4-amine, 6-(6-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl)-N-methoxyl group-N-methyl caproamide, 1-[2, 2-dimethyl-3-(mesyl) propyl group]-2-(ethoxyl methyl)-1H-imidazoles [4, 5-c] quinolin-4-amines, N-[4-(4-amino-2-methyl-1H-imidazoles [4, 5-c] quinoline-1-yl) butyl]-N-methyl-N-phenylurea, 1-{3-[4-amino-1-(2-methyl-propyl)-1H-imidazoles [4, 5-c] quinoline-8-yl] phenyl } ethyl ketone, 7-(4-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl)-2-methyl heptan-2-alcohol, N-methyl-4-(4-amino-2-ethyl-1H-imidazoles [4, 5-c] quinoline-1-yl) butane-1-sulfonamide, N-(4-methoxy-benzyl)-4-(4-amino-2-ethyl-1H-imidazoles [4, 5-c] quinoline-1-yl) butane-1-sulfonamide, N-{2-[4-amino-3-(ethoxyl methyl)-6, 7-dimethyl-1H-imidazole radicals [4, 5-c] pyridine-1-yl]-1, 1-dimethyl ethyl } Methanesulfomide, 2-ethoxymethyl-1-(3-methoxy-propyl)-7-(5-hydroxymethylpyridine-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 1-[(2, 2-dimethyl-1, 3-dioxolanes-4-yl) methyl]-2-(ethoxyl methyl)-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 4-[3-(4-amino-6, 7-dimethyl-2-propyl group-1H-imithizo[4, 5-c] pyridine-1-yl) propane-1-sulfonyl]-ethyl benzoate, 2-butyl-1-{2-[2-(methyl sulphonyl) ethyoxyl] ethyl }-1H-imidazoles [4, 5-c] quinolin-4-amines, N-(2-{4-amino-2-ethoxyl methyl-7-[6-(mesyl amino) hexyloxy]-1H-imidazoles [4, 5-c] quinoline-1-yl }-1, 1-dimethyl ethyl) Methanesulfomide, N-(6-{[4-amino-2-ethoxyl methyl-1-(2-mesyl amino-2-methyl propyl group)-1H-imidazoles [4, 5-c] quinoline-7-yl] oxygen base } hexyl) acetamide, 1-[4-(1, 1-dioxy isothiazoline-2-yl) butyl]-2-ethoxyl methyl-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 1-[4-(1, 1-dioxy isothiazoline-2-yl) butyl]-2-ethoxyl methyl-7-(pyridin-4-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 1-[4-(1, 1-dioxy isothiazoline-2-yl) butyl]-2-ethoxyl methyl-7-phenyl-1H-imidazoles [4, 5-c] quinolin-4-amines, 2-(ethoxyl methyl)-1-{[1-(methyl sulphonyl) piperidin-4-yl] methyl }-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 2-(ethoxyl methyl)-1-[(1-isobutyl group piperidin-4-yl) methyl]-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, 2-(ethoxyl methyl)-1-{[1-(morpholine-4-base carbonyl) piperidin-4-yl] methyl }-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, cyclopropanic acid [3-(4-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl) propoxyl group] amide, isopropylamino formic acid 4-amino-2-(2-methoxy ethyl)-1-propyl group-1H-imidazoles [4, 5-c] quinoline-7-base ester, 4-(4-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl) ethyl n-butyrate., 1-[4-amino-2-ethyl-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinoline-1-yl]-2-methyl propan-2-ol, 1-(4-amino-2-ethyl-7-[5-{ methylol } pyridin-3-yl]-1H-imidazoles [4, 5-c] quinoline-1-yl)-2-methyl propan-2-ol, 1-(3-[4-amino-2-(2-methoxy ethyl)-8-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinoline-1-yl] propyl group) pyrrolidin-2-one, N-(2-{4-amino-2-ethoxyl methyl-7-[6-(mesyl amino) hexyloxy]-1H-imidazoles [4, 5-c] quinoline-1-yl }-1, 1-dimethyl ethyl) acetamide, 1-{3-[4-amino-7-(3-hydroxymethyl phenyl)-2-(2-methoxy ethyl)-1H-imidazoles [4, 5-c] quinoline-1-yl] propyl group } pyrrolidin-2-one, N-{4-[4-amino-2-ethoxyl methyl-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinoline-1-yl] butyl }-N '-propyl group urea, N-{4-[4-amino-2-ethoxyl methyl-7-(pyridin-3-yl)-1H-imidazoles [4, 5-c] quinoline-1-yl] butyl } butyramide, 5-(4-amino-2-propyl group-1H-imidazoles [4, 5-c] quinoline-1-yl)-4, 4-dimethyl-penten-2-ketone, 1-cyclohexyl methyl-2-ethoxyl methyl-7-(5-hydroxymethylpyridine-3-yl)-1H-imidazoles [4, 5-c] quinolin-4-amines, N, N-dimethyl-5-(4-amino-2-ethoxyl methyl-1H-imidazoles [4, 5-c] quinoline-1-yl) pentane-1-sulfonamide, N-{3-[(4-amino-2-ethoxyl methyl-1H-imidazoles [4, 5-c] quinoline-1-yl) amino] propyl group } Methanesulfomide and/or N, N-dimethyl-4-(4-amino-2-ethoxyl methyl-1H-imidazoles [4, 5-c] quinoline-1-yl) butane-1-sulfonamide.

Other suitable TLR7 selective agonists include but not limited to 2-(ethoxyl methyl)-1-(2-methyl-propyl)-1H-imidazoles [4,5-c] quinolin-4-amines (United States Patent (USP) 5,389,640); 2-methyl isophthalic acid-[2-(3-pyridin-3-yl propoxyl group) ethyl]-1H-imidazoles [4,5-c] quinolin-4-amines (WO02/46193); N-(2-{2-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4,5-c] quinoline-1-yl] ethyoxyl } ethyl)-N-hexahydrotoluene carboxylic acid amides (United States Patent (USP) discloses 2004/0171086); 1-[2-(benzyloxy) ethyl]-2-methyl isophthalic acid H-imidazoles [4,5-c] quinolin-4-amines (WO02/46189); N-{8-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4,5-c] quinoline-1-yl] octyl group }-N-phenylurea (United States Patent (USP) discloses 2004/0171086 (IRM5)); 2-butyl-1-[5-(methyl sulphonyl) amyl group]-1H-imidazoles [4,5-c] quinolin-4-amines (WO02/46192); N-{3-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4,5-c] quinoline-1-yl] propyl group }-4-methyl benzenesulfonamide (United States Patent (USP) 6,331,539); And N-[4-(4-amino-2-ethyl-1H-imidazoles [4,5-c] quinoline-1-yl) butyl] cyclohexane extraction carboxylic acid amides (United States Patent (USP) discloses 2004/0171086 (IRM8)).Also be applicable to use be TLR7 selective agonist N-[4-(4-amino-2-ethyl-1H-imidazoles [4,5-c] quinoline-1-yl) butyl-] Methanesulfomide (Gorden etc., 2005).

TLR8 agonist. suitable TLR8 agonist comprises the natural TLR8 agonist of separation; With synthetic TLR8 agonist.The TLR8 agonist separating from natural TLR8 agonist source is generally purification, and the purity of the TLR8 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR8 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR8 agonist comprises the TLR8 agonist being not adhered on any other compound.TLR8 agonist comprises the TLR8 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR8 agonist is directly attached on another compound.In other embodiments, TLR8 agonist is attached on another compound by joint.

TLR8 agonist includes but not limited to the compound such as R-848 and derivant and analog.Suitable TLR8 agonist comprises the compound with the PA condensing with five Yuans nitrogen heterocyclic rings.This compounds comprises as imidazole quinoline amine, include but not limited to the imidazole quinoline amine replacing, the imidazole quinoline amine of the imidazole quinoline amine that the imidazole quinoline amine that for example amide replaces, the imidazole quinoline amine of sulfonamide substitutions, carbamide replace, the imidazole quinoline amine that aryl ether replaces, heterocyclic ether replacement, the imidazole quinoline amine that amino ethers replaces, the imidazole quinoline amine that sulphonyl amidogen ether replaces, the imidazole quinoline ether that carbamide replaces, the imidazole quinoline amine that thioether replaces, and the imidazole quinoline amine of 6-, 7-, 8-or 9-aryl or heteroaryl replacement; Imidazolidine quinolinamine, includes but not limited to imidazolidine quinolinamine, the imidazolidine quinolinamine of sulfonamide substitutions, the imidazolidine quinolinamine of carbamide replacement, the imidazolidine quinolinamine that aryl ether replaces, the imidazolidine quinolinamine that heterocyclic ether replaces, the imidazolidine quinolinamine that amino ethers replaces, the imidazolidine quinolinamine that sulphonyl amidogen ether replaces, the imidazolidine quinoline ether of carbamide replacement and the imidazolidine quinolinamine that thioether replaces that amide replaces; Imidazopyridine amine, includes but not limited to imidazopyridine amine, the imidazopyridine amine of sulfonamide substitutions, the imidazopyridine amine of carbamide replacement, the imidazopyridine amine that aryl ether replaces, the imidazopyridine amine that heterocyclic ether replaces, the imidazopyridine amine that amino ethers replaces, the imidazopyridine amine that sulphonyl amidogen ether replaces, the imidazopyridine ether of carbamide replacement and the imidazopyridine amine that thioether replaces that amide replaces; The imidazole quinoline amine of 1,2-bridge joint; The cycloalkyl imidazopyridine amine that 6,7-condenses; Imidazo naphthyridines amine; Imidazolidine naphthyridines amine;

azoles quinolinamine; Thiazole quinolinamine;

azoles pyridine amine; Thiazole pyridine amine;

azoles naphthyridines amine; Thiazole naphthyridines amine; With the 1H-imidazoles dimer, quinolinamine, tetrahydroquinoline amine, naphthyridines amine or the Tetrahydronaphthyridderivates amine that condense with pyridine amine.

In a specific embodiments, TLR8 agonist is the imidazole quinoline amine that amide replaces.In an alternative embodiment, TLR8 agonist is the imidazole quinoline amine of sulfonamide substitutions.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that carbamide replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that aryl ether replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that heterocyclic ether replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that amino ethers replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that sulfanilamide ether replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline ether that carbamide replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that thioether replaces.In another alternative embodiment, TLR8 agonist is the imidazole quinoline amine that 6-, 7-, 8-or 9-aryl or heteroaryl replace.

In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that amide replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine of sulfonamide substitutions.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that carbamide replaces.

In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that aryl ether replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that heterocyclic ether replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that amino ethers replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that sulphonyl amidogen ether replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinoline ether that carbamide replaces.In another alternative embodiment, TLR8 agonist is the imidazolidine quinolinamine that thioether replaces.

In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that amide replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine of sulfonamide substitutions.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that carbamide replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that aryl ether replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that heterocyclic ether replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that amino ethers replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that sulphonyl amidogen ether replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine ether that carbamide replaces.In another alternative embodiment, TLR8 agonist is the imidazopyridine amine that thioether replaces.

In another alternative embodiment, TLR8 agonist is 1,2-bridge joint imidazole quinoline amine.In another alternative embodiment, TLR8 agonist is the cycloalkyl imidazopyridine amine that 6,7-condenses.

In another alternative embodiment, TLR8 agonist is imidazo naphthyridines amine.In another alternative embodiment, TLR8 agonist is imidazolidine naphthyridines amine.In another alternative embodiment, TLR8 agonist is

azoles quinolinamine.In another alternative embodiment, TLR8 agonist is thiazole quinolinamine.In another alternative embodiment, TLR8 agonist is azoles pyridine amine.In another alternative embodiment, TLR8 agonist is thiazole pyridine amine.In another alternative embodiment, TLR8 agonist is

azoles naphthyridines amine.In another alternative embodiment, TLR8 agonist is thiazole naphthyridines amine.

In another alternative embodiment, TLR8 agonist is 1H-imidazoles dimer, quinolinamine, tetrahydroquinoline amine, naphthyridines amine or the Tetrahydronaphthyridderivates amine condensing with pyridine amine.

In some embodiments, TLR8 agonist is selectivity TLR8 agonist, and for example described agonist regulates cytoactive by TLR8, but does not regulate cytoactive by TLR7.TLR8 selective agonist is included in United States Patent (USP) and discloses those that show in 2004/0171086.This type of TLR8 selective agonist compound includes but not limited to that United States Patent (USP) discloses the compound showing in 2004/0171086, it comprises N-{4-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4,5-c] quinoline-1-yl] butyl } quinoline-3-carboxylic acid amides, N-{4-[4-amino-2-(2-methoxy ethyl)-1H-imidazoles [4,5-c] quinoline-1-yl] butyl } quinoxaline-2-carboxylic acid amides and N-[4-(4-amino-2-propyl group-1H-imidazoles [4,5-c] quinoline-1-yl) butyl] morpholine-4-carboxylic acid amides.

Other suitable TLR8 selective agonists include but not limited to 2-propyl group thiazole [4,5-c] quinolin-4-amines (United States Patent (USP) 6,110,929); N ¹-[2-(4-amino-2-butyl-1H-imidazoles [4,5-c] [1,5] naphthyridines-1-yl) ethyl]-2-amino-4-methylpent amide (United States Patent (USP) 6,194,425); N ¹-[4-(4-amino-1H-imidazoles [4,5-c] quinoline-1-yl) butyl]-2-phenoxy group-Benzoylamide (United States Patent (USP) 6,451,810); N ¹-[2-(4-amino-2-butyl-1H-imidazoles [4,5-c] quinoline-1-yl) ethyl]-1-the third sulfonamide (United States Patent (USP) 6,331,539); N-{2-[2-(4-amino-2-ethyl-1H-imidazoles [4,5-c] quinoline-1-yl) ethyoxyl] ethyl }-N '-phenylurea (United States Patent (USP) discloses 2004/0171086); 1-{4-[3,5-Dichlorobenzene base] sulfenyl } butyl)-2-ethyl-1H-imidazoles [4,5-c] quinolin-4-amines (United States Patent (USP) discloses 2004/0171086); N-{2-[4-amino-2-(ethoxyl methyl)-1H-imidazoles [4,5-c] quinoline-1-yl] ethyl }-N '-(3-cyano-phenyl) carbamide (WO00/76518 and U.S. Patent Publication No. 2004/0171086); With 4-amino-α, alpha-alpha-dimethyl-2-methoxy ethyl-1H-imidazoles [4,5-c] quinoline-1-ethanol (United States Patent (USP) 5,389,640).As the compound in U.S. Patent Publication No. 2004/0171086 that comprises of TLR8 selective agonist.What be also applicable to using is compound 2-propyl group thiazole-4,5-c) quinolin-4-amines (Gorden etc., 2005 is the same).

TLR9 agonist. suitable TLR9 agonist comprises the natural TLR9 agonist of separation; With synthetic TLR9 agonist.The TLR9 agonist separating from natural TLR9 agonist source is generally purification, and the purity of the TLR9 agonist of for example purification is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.Synthetic TLR9 agonist is prepared by standard method, and purity is generally at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher than 99%.

TLR9 agonist comprises the TLR9 agonist being not adhered on any other compound.TLR9 agonist comprises the TLR9 agonist being covalently or non-covalently attached on another compound.In some embodiments, TLR9 agonist is directly attached on another compound.In other embodiments, TLR9 agonist is attached on another compound by joint.

The example of TLR9 agonist (herein also referred to as " TLR9 part ") comprises such nucleic acid, and it comprises sequence 5 '-CG-3 ' (" CpG nucleic acid "), and C is unmethylated in some aspects.The term " polynucleotide " that exchange is used in the context of TLR9 ligand molecular and " nucleic acid " refer to the polynucleotide of any length, comprise strand and double chain oligonucleotide (comprise deoxyribonucleotide, ribonucleotide or both), oligonucleotide and the oligonucleoside etc. modified, they are independent, or as a large nucleic acids construct part more, or as with the part of the conjugate of non-nucleic acid molecules (as polypeptide).Therefore, TLR9 part can be for example single stranded DNA (ssDNA), double-stranded DNA (dsDNA), single stranded RNA (ssRNA) or double-stranded RNA (dsRNA).TLR9 part also comprises the rough antibacterial through detoxification (for example mycobacteria) RNA or DNA, and the enrichment plasmid of enrichment TLR9 part.In some embodiments, " plasmid of enrichment TLR9 part " refers to linearity or circular plasmids, and it comprises (or comprising through transformation) than the CpG motif of the greater number of conventionally finding in mammalian DNA.

The limiting examples of the plasmid of enrichment TLR9 part has been described in (1997) such as Roman.The modification of oligonucleotide includes but not limited to the modification of 3 ' OH or 5 ' OH group, modification, the modification of saccharic composition and the modification of phosphate group of nucleotide base.

TLR9 part can comprise at least one nucleoside (it comprises L-sugar).L-sugar can be deoxyribose, ribose, pentose, deoxypentose, hexose, deoxyhexamethylose, glucose, galactose, arabinose, xylose, lyxose or sugar " analog " cyclopenta.L-sugar can be pyrans glycosyl or furyl glycosyl form.

The expression of any aminoacid sequence of (also not needing them to provide) polynucleotide encoding is not generally provided TLR9 part, and therefore the sequence of TLR9 part can be that (and normally) is noncoding.TLR9 part can comprise linear two strands or single chain molecule, ring molecule, maybe can comprise linearity and circular segments.TLR9 part can be strand, can be to be maybe double-stranded wholly or in part.

In some embodiments, TLR9 part for the inventive method is oligonucleotide, for example, be made up of to the sequence of approximately 7 nucleotide to approximately 10 nucleotide, approximately 5 nucleotide to approximately 15 nucleotide, approximately 5 nucleotide to approximately 30 nucleotide, approximately 5 nucleotide to approximately 25 nucleotide, 20 nucleotide to approximately 50 nucleotide, approximately 15 nucleotide to approximately 100 nucleotide, approximately 12 nucleotide to approximately 200 nucleotide, approximately 10 nucleotide approximately 5 nucleotide of length.In some embodiments, length is less than approximately 15 nucleotide, is less than approximately 12 nucleotide, is less than approximately 10 nucleotide or is less than the TLR9 part of approximately 8 nucleotide and larger point sub-connection.

In some embodiments, TLR9 part does not provide the expression of peptide in eukaryotic cell or polypeptide, for example, in eukaryotic cell, draw the generation that TLR9 part does not cause peptide or polypeptide, because TLR9 part does not provide the transcribing of mRNA of encoded peptide or polypeptide.In these embodiments, TLR9 part lacks the required promoter region of eukaryotic cell transcription and other control elements.

TLR9 part can separate from antibacterial, for example, separate from bacterial origin; Produce (for example producing by the standard method of chemosynthesis polynucleotide) by synthetic method; Produce by standard recombination method, then separate from bacterial origin; Or the combination of said method.In many embodiments, TLR9 part is purification, and for example purity is at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or higher, for example 99.5%, 99.9% or higher.In many embodiments, TLR9 part is then purification of chemosynthesis.

In other embodiments, TLR9 part is the part of larger constructs (for example plasmid vector, viral vector or other this type of constructs).A large amount of plasmids and viral vector are known in the art, need to not describe in detail herein., at multiple publication, comprise for example Current Protocols in Molecular Biology, in (1987 and upgrade), described a large amount of examples of such carriers.

Generally speaking, comprise at least one unmethylated CpG motif for the TLR9 part of the present composition.For example, in some mammalian species (, Rodents), in polynucleotide, the relative position of any CpG sequence is 5 '-CG-3 ' (that is, comparatively speaking, C is in 5 ' position, and G is in 3 ' position).

In some embodiments, TLR9 part comprises central palindrome core sequence (it comprises at least one CpG sequence), wherein said central palindrome core sequence contains phosphodiester backbone, and the flank of wherein said palindrome core sequence one or both sides is the poly guanosine sequence containing phosphorothioate backbone.

In other embodiments, TLR9 part comprises one or more TCG sequences near nucleic acid 5 ' end or its; At least two extra CG dinucleotide.In some this embodiments, described at least two extra CG dinucleotide are separated by three nucleotide, two nucleotide or a nucleotide.In some this embodiments, described at least two extra CG dinucleotide are adjacent to each other.In some this embodiments, TLR9 part comprises (TCG) n, wherein n=1 to 3 at 5 ' end of nucleic acid.In other embodiments, TLR9 part comprises (TCG) n, wherein n=1 to 3, and wherein (TCG) n sequence has a nucleotide, two nucleotide, three nucleotide, four nucleotide or five nucleotide at 5 ' end flank of (TCG) n sequence.

Exemplary total CpG motif for TLR9 part of the present invention includes but not limited to: 5 '-purine-purine-(C)-(G)-pyrimidine-pyrimidine-3 ', and wherein TLR9 part comprises the CpG motif that flank is at least two purine nucleotides (such as GG, GA, AG, AA, II etc.) and at least two pyrimidine nucleotides (CC, TT, CT, TC, UU etc.); 5 '-purine-TCG-pyrimidine-pyrimidine-3 '; 5 '-TCG-N-N-3 '; Wherein N is any base; 5 '-Nx (CG) nNy, wherein N is any base, wherein x and y are any integer of 0 to 200 independently, for example 0,1,2,3,4,5,6,7,8,9,10,11-15,16-20,21-25,25-30,30-50,50-75,75-100,100-150 or 150-200; And n is 1 or larger any integer, for example 1,2,3,4,5,6,7,8,9,10 or larger.5 '-Nx (TCG) nNy, wherein N is any base, wherein x and y are any integer of 0 to 200 independently, for example 0,1,2,3,4,5,6,7,8,9,10,11-15,16-20,21-25,25-30,30-50,50-75,75-100,100-150 or 150-200; And n is 1 or larger any integer, for example 1,2,3,4,5,6,7,8,9,10 or larger.5 '-(TCG) n-3 ', wherein n is 1 or larger any integer, for example with provide TLR9 part based on TCG (for example, wherein n=3, described polynucleotide comprise sequence 5 '-TCGNNTCGNNTCG-3 '; SEQ ID NO:3); 5 ' Nm-(TCG) n-Np-3 ', wherein N is any nucleotide, and wherein m is 0,1,2 or 3, and wherein n is 1 or larger any integer, and wherein p is 1,2,3 or 4; 5 ' Nm-(TCG) n-Np-3 ', wherein N is any nucleotide, wherein m is 0 to 5, and wherein n is 1 or larger any integer, wherein p is 4 or larger, and wherein sequence N-N-N-N comprises and is adjacent to each other or by a nucleotide, two nucleotide or three separated at least two CG dinucleotide of nucleotide; With 5 '-purine-purine-CG-pyrimidine-TCG-3 '.

In the time of sequence that nucleic acid TLR9 part comprises following general formula: 5 '-Nm-(TCG) n-Np-3 ', wherein N is any nucleotide, wherein m is 0 to 5, and wherein n is 1 or larger any integer, wherein p is 4 or larger, and wherein sequence N-N-N-N comprises and is adjacent to each other or by a nucleotide, two nucleotide or three separated at least two CG dinucleotide of nucleotide, can be used for example T LR9 part of the present invention includes but not limited to: the sequence of (1) this general formula, wherein n=2, Np is NNCGNNCG; (2) sequence of this general formula, wherein n=2, Np is AACGTTCG; (3) sequence of this general formula, wherein n=2, Np is TTCGAACG; (4) sequence of this general formula, wherein n=2, Np is TACGrACG; (5) sequence of this general formula, wherein n=2, Np is ATCGATCG; (6) sequence of this general formula, wherein n=2, Np is CGCGCGCG; (7) sequence of this general formula, wherein n=2, Np is GCCGGCCG; (8) sequence of this general formula, wherein n=2, Np is CCCGGGCG; (9) sequence of this general formula, wherein n=2, Np is GGCGCCCG; (10) sequence of this general formula, wherein n=2, Np is CCCGTTCG; (11) sequence of this general formula, wherein n=2, Np is GGCGTTCG; (12) sequence of this general formula, wherein n=2, Np is TTCGCCCG; (13) sequence of this general formula, wherein n=2, Np is TTCGGGCG; (14) sequence of this general formula, wherein n=2, Np is AACGCCCG; (15) sequence of this general formula, wherein n=2, Np is AACGGGCG; (16) sequence of this general formula, wherein n=2, Np is CCCGAACG; (17) sequence of this general formula, wherein n=2, Np is GGCGAACG; And wherein, at 1-17 in any one, m=0,1,2 or 3.

In the time of sequence that nucleic acid TLR9 part comprises following general formula: 5 '-Nm-(TCG) n-Np-3 ', wherein N is any nucleotide, wherein m is 0,1,2 or 3, wherein n is 1 or larger any integer, and wherein p is 1,2,3 or 4, can be used for example T LR9 part of the present invention and include but not limited to: the sequence of (1) this general formula, wherein m=0, n=1, and Np is T-T-T; (2) sequence of this general formula, wherein m=0, n=1, and Np is T-T-T-T; (3) sequence of this general formula, wherein m=0, n=1, and Np is C-C-C-C; (4) sequence of this general formula, wherein m=0, n=1, and Np is A-A-A-A; (5) sequence of this general formula, wherein m=0, n=1, and Np is A-G-A-T; (6) sequence of this general formula, wherein Nm is T, n=1, and Np is T-T-T; (7) sequence of this general formula, wherein Nm is A, n=1, and Np is T-T-T; (8) sequence of this general formula, wherein Nm is C, n=1, and Np is T-T-T; (9) sequence of this general formula, wherein Nm is G, n=1, and Np is T-T-T; (10) sequence of this general formula, wherein Nm is T, n=1, and Np is A-T-T; (11) sequence of this general formula, wherein Nm is A, n=1, and Np is A-T-T; (12) sequence of this general formula, wherein Nm is C, n=1, and Np is A-T-T.

Can be used for the core texture upstream of TLR9 part of the present invention and/or the flank in downstream can be nucleotide or the nucleoside of any quantity or composition.In some embodiments, the core sequence length of TLR9 part is at least 6 bases or 8 bases, and the length of complete TLR9 part (core sequence adds 5 ', 3 ' or both flanking sequence) generally in 6 bases or 8 bases between up to approximately 200 bases.

Can be used for the TLR9 part based on DNA of the present invention and include but not limited to such polynucleotide, it comprises one or more following nucleotide sequences:

AGCGCT, AGCGCC, AGCGTT, AGCGTC, AACGCT, AACGCC, AACGTT, AACGTC, GGCGCT, GGCGCC, GGCGTT, GGCGTC, GACGCT, GACGCC, GACGTT, GACGTC, GTCGTC, GTCGCT, GTCGrT, GTCGCC, ATCGTC, ATCGCT, ATCGTT, ATCGCC, TCGTCG, and TCGTCGTCG.

Include but not limited to such polynucleotide for other TLR9 parts of the present invention, it comprises one or more following nucleotide sequences:

TCGXXXX, TCGAXXX, XTCGXXX, XTCGAXX, TCGTCGA, TCGACGT, TCGAACG, TCGAGAT, TCGACTC, TCGAGCG, TCGATTT, TCGCTTT, TCGGTTT, TCGTTTT, TCGTCGT, ATCGATT, TTCGTTT, TTCGATT, ACGTTCG, AACGTTC, TGACGTT, TGTCGTT, TCGXXX, TCGAXX, TCGTCG, AACGTT, ATCGAT, GTCGTT, GACGTT, TCGXX, TCGAX, TCGAT, TCGTT, TCGTC, TCGA, TCGT, TCGX, and TCG

(wherein " X " is any nucleotide).

Can be used for the TLR9 part based on DNA of the present invention and include but not limited to such polynucleotide, it comprises one or more following eight aggressiveness nucleotide sequences:

AGCGCTCG, AGCGCCCG, AGCGTTCG, AGCGTCCG, AACGCTCG, AACGCCCG, AACGTTCG, AACGTCCG, GGCGCTCG, GGCGCCCG, GGCGTTCG, GGCGTCCG, GACGCTCG, GACGCCCG, GACGTTCG, and GACGTCCG.

The TLR9 part that can be used for implementing the inventive method can comprise one or more above-mentioned any CpG motifs.For example, can be used for TLR9 part of the present invention and can comprise the same CpG motif that one or many (for example 2,3,4,5 or more times) occurs.Or, TLR9 part (for example can comprise multiple CpG motifs, 2,3,4,5 or more), at least two in wherein said multiple CpG motifs have different consensus sequences, or all CpG motifs in described TLR9 part have different consensus sequences.

Can be used for TLR9 part of the present invention and can comprise or not comprise palindrome district.If existed, palindrome only extends to (if existed, in core six aggressiveness or eight aggressiveness sequences) in CpG motif, maybe can comprise more six aggressiveness or eight aggressiveness sequences and flanking nucleotide sequence.

Polymer TLR9 part.In some embodiments, TLR9 part is polymer.Polymer TLR9 part comprises 2,3,4,5,6,7,8,9,10 or the above-mentioned nucleic acid TLR9 of more body (monomer) part, it is connected, is connected by covalent bond by non-covalent bond, is connected to each other directly or connects by one or more spacers.Suitable spacer comprises nucleic acid and non-nucleic acid molecules, as long as they are biocompatible.In some embodiments, the linear array that polymer TLR9 part comprises monomer TLR9 part.In other embodiments, polymer TLR9 part is branch-like or the dendroid array of monomer TLR9 part.

In some embodiments, polymer TLR9 part has general structure (X1) n (X2) n, wherein X is nucleic acid TLR9 part as above, and has following length: from approximately 6 nucleotide to approximately 200 nucleotide, for example, from approximately 6 nucleotide to approximately 8 nucleotide, from approximately 8 nucleotide to approximately 10 nucleotide, from approximately 10 nucleotide to approximately 12 nucleotide, from approximately 12 nucleotide to approximately 15 nucleotide, from approximately 15 nucleotide to approximately 20 nucleotide, from approximately 20 nucleotide to approximately 25 nucleotide, from approximately 25 nucleotide to approximately 30 nucleotide, from approximately 30 nucleotide to approximately 40 nucleotide, from approximately 40 nucleotide to approximately 50 nucleotide, from approximately 50 nucleotide to approximately 60 nucleotide, from approximately 60 nucleotide to approximately 70 nucleotide, from approximately 70 nucleotide to approximately 80 nucleotide, from approximately 80 nucleotide to approximately 90 nucleotide, from approximately 90 nucleotide to approximately 100 nucleotide, from approximately 100 nucleotide to approximately 125 nucleotide, from approximately 125 nucleotide to approximately 150 nucleotide, from approximately 150 nucleotide to approximately 175 nucleotide or from 175 nucleotide to approximately 200 nucleotide, and wherein n is any numerical value of from 1 to approximately 100, for example n=1,2,3,4,5,6,7,8,9,10, from 10 to approximately 15, from 15 to approximately 20, from 20 to approximately 25, from 25 to approximately 30, from 30 to approximately 40, from 40 to approximately 50, from 50 to approximately 60, from 60 to approximately 70, from 70 to approximately 80, from 80 to approximately 90 or from 90 to approximately 100.In these embodiments, X and X2 list the difference of at least one nucleotide each other at nucleotides sequence, and list the difference that can have 2,3,4,5,6,7,8,9,10 or more bases at nucleotides sequence each other.

As noted above, in some embodiments, polymer TLR9 part of the present invention comprises the separated TLR9 part of TLR9 part with vicinity by spacer.In some embodiments, spacer is non-TLR9 ligand nucleic acid.In other embodiments, spacer is non-nucleic acid moiety.Suitable spacer is included in United States Patent (USP) and discloses those that describe in 20030225016.Use any known method to make TLR9 part generation multimerization.

TLR9 is ligand modified.Can modify in many ways the TLR9 part that is applicable to the present composition.For example, TLR9 part can comprise skeleton phosphate group and (for example modify, between methyl phosphorodithioate, thiophosphate, phosphoramidate (phosphoroamidate) and phosphorodithioate nucleotide, be connected), described modification for example can strengthen its body internal stability, makes them particularly useful in treatment application.It is thiophosphate or the phosphordithiic acid ester-formin that changes into nucleic acid TLR9 part that useful especially phosphate group is modified.Thiophosphate and phosphorodithioate have higher vivo degradation resistance than the oligonucleotide homologue of its unmodified, have improved the half-life of TLR9 part and have made their availabilities in patient higher.

The TLR9 part of other modifications that the present invention includes be included in 5 ' end, 3 ' end or 5 ' and 3 ' end there is the TLR9 part of modification.For example, 5 ' and/or 3 ' end is binding molecule (nucleic acid, non-nucleic acid or both) covalently or non-covalently, thereby for example improves the bioavailability of TLR9 part, improve while needing and take in efficiency, promote to sending of object cell etc.The molecule that is used for puting together with TLR9 part (for example includes but not limited to cholesterol, phospholipid, fatty acid, sterin, oligosaccharide, polypeptide, immunoglobulin), peptide, antigen (for example, peptide, micromolecule etc.), linearity or ringed nucleus acid molecule (for example, plasmid), insoluble holder, therapeutical peptide etc.The therapeutical peptide that is suitable for being attached on TLR9 agonist includes but not limited to dendritic cell somatomedin (for example, GM-CSF); Cytokine; Interferon (for example, IFN-α and IFN-β etc.); TNF-alpha-2 antagonists etc.

In some embodiments, TLR9 part is connected (for example, put together, covalently bound, non-covalent connection or be adsorbed onto on it) with insoluble holder.The exemplary unrestricted example of insoluble holder is cation poly-(D, L-lactide-co-Acetic acid, hydroxy-, bimol. cyclic ester).

Other TLR9 ligand conjugates and production method thereof are known in the art, and are described in for example WO98/16427 and WO98/55495.Therefore, term " TLR9 part " comprises such conjugate, and it comprises nucleic acid TLR9 part.

Polypeptide (for example therapeutical peptide) can directly be puted together or for example indirectly be conjugated on TLR9 part by linkers.Multiple linkers is known in the art, and can be used in conjugate.Peptide can be brought in realization by N end or the C of the reactive side chain of peptide or peptide with being connected of oligonucleotide.Oligonucleotide can be at 3 ' or 5 ' end with being connected of peptide, or middle.Joint can be organic, inorganic or half organic molecule, and can be organic molecule polymer, inorganic molecule or the copolymer that comprises inorganic and organic molecule.

If existed, the length of linkers is generally enough to allow the polypeptide of oligonucleotide and/or polynucleotide and connection that certain flexibility occurs between described oligonucleotide and polypeptide to move.The length of linkers is generally an about 6-50 atom.Linkers can be also for example aryl ethane, the ethylene glycol oligomer that contains 2-10 monomeric unit, diamidogen, diacid, aminoacid or its combination.Also can use other linkers that can be combined with oligonucleotide based on present disclosure.

B.NOD sample receptor (NLR) agonist

NOD sample receptor (NLR) is the cytoplasmic protein can in the adjusting of inflammatory and apoptotic responses with several functions.About 20 this protein in mammalian genes group, are found, and comprise two main subfamilies, II class MHC transactivator (CIITA) and some other molecules (for example, IPAF and BIRC1) of being called NOD and NALP.Current understanding proposes, some in these protein are identified endogenous molecule or microorganism molecule or stress response, and (for example form activation inflammatory Caspase, Caspase 1) oligomer of (it causes that important inflammatory cytokine is as the cutting of IL-1 and activation), and/or activate NF-κ B signal pathway, to induce the generation of inflammatory molecule.There are some different names in NLR family, comprises CATERPILLER (or CLR) or NOD-LRR family.

The part of current known NOD1 and NOD2.NOD1 identification is called the molecule of meso DAP, and it is only the Peptidoglycan component of gram negative bacteria.MDP (muramyldipeptide) in NOD2 albumen identification born of the same parents, it is the Peptidoglycan component of Gram-positive and negative bacterium.NODS carries out signal transduction by serine-threonine kinase (being called RIP2) in NF-κ B and map kinase approach.The name of NOD albumen is because they contain the nucleotide of being combined with nucleotide triphosphoric acid in conjunction with oligomerization domain.NOD holds CARD domain to carry out signal transduction by N, to activate downstream gene induction event, and holds rich leucine to repeat (1eucin-rich repeat, LRR) district and microorganism interaction of molecules by C.

As NOD, the LRR that NALP albumen contains C end, it be it seems and work as Regulatory domain, and can participate in the identification of microbial pathogens.Same as NOD, these protein also contain the nucleotide binding site (NBS) of nucleotide triphosphoric acid.Hold the interaction of the mediation of pyrin (PYD) domain and other protein (for example adapter molecule ASC) by N.In people, this subfamily has 14 members (being called NALP1 to NALP14).The sudden change of NALP3 causes cold self inflammatory syndrome of self inflammatory diseases familial, hereditary familial urticaria syndrome and neonate morbidity multisystem inflammatory diseases.The activation attached bag of NALP3 is drawn together muramyldipeptide, DNA of bacteria, ATP, toxin, double-stranded RNA, paramyxovirus and uric acid crystal.

Also shown that other NLR respond to Salmonella and Legionnella and activate Caspase 1 as IPAF and NAIP5/Birc1e.

NLR agonist includes but not limited to GM tripeptides (shigella flexneri (Shigella flexneri)), meso L-lanthionine (helicobacter pylori), meso DAP, γ-D-Glu-DAP (iEDAP) (enteroinvasive E.Coli), D-lactyl-L-ala-y-Glu-meso-DAP-Gly (FK156) (pseudomonas), heptanoyl group-γ-Glu-meso-DAP-D-ala (FK565) (chlamydia (Chlamydia), monokaryon Listerella (Listeria monocyotgenes)), MDP (monokaryon Listerella), MurNAc-L-Ala-γ-D-Glu-L-Lys (M-TRILys) (streptococcus pneumoniae, Salmonella typhimurium, the little flagellin of Salmonella flexner, bacteria RNA, ATP, nigericin (Nigericin), Maitotoxin, uric acid crystal, aerolysin (Aerolysin) and anthrax lethal toxin.

C.RIG sample receptor (RLR) agonist

The interior various kinds of cell of body can experience infectious virus and startup is referred to as the congenital reaction of replying of antiviral.These are replied and comprise and produce antiviral cell factor (as I type interferon (IFN)), synthetic disease-resistant toxenzyme subsequently, and it is responsible for break virus and copies and promote adaptive immune response.The impression of RIG (tretinoin inducible genes) sample receptor triggers the viral RNA molecule of innate immune system component.The part of RLR includes but not limited to ssRNA, dsRNA, poly-inosine-POLY CYTIDYLIC ACID (synthetic analogues of " poly (rI:rC) ", double-stranded RNA (dsRNA) and other viral nucleic acids and analog thereof, comprise part rna virus cdna group (for example, Japanese encephalitis virus (JEV), vesicular stomatitis virus (VSV), influenza virus, dengue virus, west nile virus, reovirus and encephalomyocarditis virus (EMCV)).RNA section or analog can be at least 20,25,30,35,40 or more nucleotide or nucleotide pair or equivalent.In some aspects, RNA is 5 ' triphosphoric acid RNA.

D. leukocytic immunity globulin sample receptor (Leukocyte Immunoglobulin-Like receptor, LIR) agonist

The clone of eight kinds of LIR-1 correlation molecules (consult Fanger etc., 1999 and list of references wherein) (having the aminoacid homogeneity of 63-84% with LIR-1) has set up a new family (LIR) of immunity receptor.LIR can divide into groups according to its structure.Five kinds of LIR (1,2,3,5 and 8) have cytoplasmic structure territory, it contains 2,3 or 4 inhibitory motifs (Immunoreceptor tyrosine-based inhibitory motif, ITIM) sample sequences based on immunity receptor tyrosine.Although two (No. 2 and No. 3 motifs in the motif based on tyrosine; I/VxYxxL/V) mate with original ITIM consensus sequence, but some in these LIR contain the motif based on tyrosine, wherein asparagine residue (No. 1 motif; Or serine residue (No. 4 motifs NxYxxL/V); SxYxxL/V) be positioned at aminoacid place, two of described tyrosine upstreams.Different from the LIR that contains ITIM, three kinds of LIR (6a, 6b and 7) contain short cytoplasmic region, and in membrane spaning domain, contain the arginine residues of positively charged.

The member of LIR family is in conjunction with I class MHC molecule.LIR-1 and LIR-2 identification HLA-A (A0101, A0301), HLA-B (B0702, B0802, B1501, B2702) and HLA-C (C0304) allele and atypia I quasi-molecule HLA-G1.Therefore the binding specificity of LIR-1 and LIR-2 is different from the binding specificity of KIR, and the latter identifies relatively limited I class MHC allele subgroup and CD94/NKG2A.Rear a part identification HLA-E, its binding pocket is occupied by the peptide of the signal sequence from specificity I class MHC antigen.

2. microbial components

a.EF2505

In some aspects, relate to treatment in suffering from infected by microbes or having the individuality of the risk that this infection occurs, suppress or alleviate the method for infected by microbes, described method comprises StIR peptide from effective dose to described individuality that use, for example enterococcus faecalis (Enterococcus faecalis) albumen EF2505 (SEQ ID NO:1) or its fragment or derivant.Conventionally, described individuality or experimenter have contacted pathogenic microbes or have had the danger of this contact.In some aspects, StIR peptide is polypeptide or peptide purification or that separate.Term " purification " or " separation " represent from other protein, to separate before component or purification out, and the purity of described component before being formulated in compositions is at least about 70,75,80,90,95,97 or 99%.In certain embodiments, the purity of component purification or that separate is for approximately or at least about 95,96,97,98,99,99.1,99.2,99.3,99.4,99.5% or higher, or can be from any scope of derivation wherein.The component of this purification then can be mixed with other components, to form compositions as described herein.

At least, at the most or approximately 5,10,15,20,25,30,35,40,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1500,1600 or 1651 continuous amino acids that restructuring StIR albumen (for example EF2505) or its fragment or section or its analog comprise SEQ ID NO:1, comprise all values and scope therebetween.In some aspects, its fragment or analog comprise at least or at the most or approximately such aminoacid sequence: it is from 1 of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120 amino acids to 100, 150, 200, 250, 300, 350, 355, 360, 365, 370, 375, 380, 390, 395, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450 amino acids, comprise all values and scope therebetween.On the other hand, its polypeptide fragment or analog include but not limited to such aminoacid sequence, at least, at the most or approximately 20,21,22,23,24,25,26,27,28,29,30 amino acids to 440,441,442,443,444,445,446,447,448,449,450 amino acids that it comprises SEQ ID NO:1.In some aspects, its polypeptide section or fragment or analog include but not limited to such aminoacid sequence, its comprise SEQ ID NO:1 at least or at the most or approximately 28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,200,250 amino acids to 440,441,442,443,444,445,446,447,448,449,450 amino acids, comprise all values and scope therebetween.More on the one hand, the aminoacid sequence that its polypeptide fragment or analog comprise comprises with 28 to 449,28 to 442,111 to 449,111 to 442,223 to 449 or 223 to 442 amino acids of SEQ ID NO:1 and has at least 70,75,80,85,90,95,96,97,98,99 or the aminoacid sequence of 100% homogeneity, comprises all values and scope therebetween.The derivant of StIR protein or its section or variant comprise insertion, disappearance and point mutation.A kind of specific insertion mutation is fusion rotein, and it comprises the aminoacid sequence for EF2505 protein external source at c-terminus or aminoterminal.

In some aspects, StIR protein or its fragment or section or derivant are used in spraying or aerosol formulations.Can be by sucking or air-breathing applying said compositions.The fragment of StIR or derivatives thereof can approximately 0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,35,40,45,50,55,60,65,70 μ g or mg/kg use to the amount of approximately 55,60,65,70,75,80,85,90,95,100,125,150,200 μ g or mg/kg whose body weight.Aspect other, can use to experimenter StIR polypeptide or peptide or its variant or derivant or the analog of approximately 0.01,0.05,0.1,0.5,1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,200 μ g or mg.Based on following discloses content, those skilled in the art can easily determine useful section, fragment or the derivant of StIR polypeptide (for example enterococcus faecalis albumen EF2505).One preferred aspect, the sequence of fragment, section or derivant and SEQ ID NO:1 has at least 75% homogeneity.On the other hand, the sequence of fragment, section or derivant and SEQ ID NO:1 has at least 80% homogeneity.On the other hand, the sequence of fragment, section or derivant and SEQ ID NO:1 has at least 85% homogeneity.On the other hand, the sequence of fragment, section or derivant and SEQ ID NO:1 has at least 90% homogeneity.On the other hand, the sequence of fragment, section or derivant and SEQ ID NO:1 has at least 95% homogeneity.

In yet another embodiment, the present invention relates to pharmaceutically acceptable compositions, it comprises one or more StIR polypeptide (for example, enterococcus faecalis albumen EF2505) or its fragment or section or derivant or analog; Antiinflammatory; Antimicrobial; And/or one or more pharmaceutical excipients.Conventionally, such composition is aseptic and does not basically contain pathogenic microbes.

B. flagellin

In some aspects, StIR compositions comprises flagellin polypeptide or its section or derivant, 10,11,12,13,14,15,16,17,18,19,20,21 or 22 continuous amino acids that it comprises the peptide QRLSTGSRINSAKDDAAGLQIA (SEQ ID NO:2) that is called LTR5 agonist.Polypeptide of the present invention also can comprise the aminoacid sequence with SEQ ID NO:2 with at least 70,80 or 90% (comprising all values and scope therebetween) homogeneity.Aspect other, flagellin is flagellin polypeptide or peptide that synthesize and/or purification or that separate.Term " purification " or " separation " represent component be before from other protein or synthetic agent or by-product isolated or purified, and the purity of described component before being formulated in compositions is at least about 95%.In certain embodiments, the purity of component purification or that separate is for approximately or at least about 80,95,96,97,98,99,99.1,99.2,99.3,99.4,99.5% or higher, or any scope that wherein can derive.Then can be by mixed to the component of this purification and other components, to form compositions as herein described.

5,10,15,20,21,22,23,24,25,30,35,40,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350 or 400 continuous amino acids (comprising all values and scope therebetween) that restructuring flagellin or its fragment or section comprise SEQ ID NO:2 or other flagellum polypeptide.These fragments or section and SEQ ID NO:2 or other flagellum polypeptide have at least, at the most or approximately 70,75,80,85,90,95,96,97,98,99 or 100% homogeneity.In some aspects, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 75% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 80% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 85% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 90% homogeneity.On the other hand, the sequence of flagellin polypeptide or section and SEQ ID NO:2 has at least 95% homogeneity.The derivant of flagellin or its section or variant comprise insertion, disappearance and the point mutation of SEQ ID NO:2.A kind of specific insertion mutation is fusion rotein, and it comprises the aminoacid sequence for flagellin external source at c-terminus or aminoterminal.Many flagellins are known in the art, include but not limited to have accession number BAB58984 (gi|14278896); YP_001330159 (gi|150402865); YP_001323483 (gi|150399716); CAA28975 (gi|1333716); CAA02137 (gi|1567895); CAA67105 (gi|1580779); AAR10473 (gi|38049688); CAR58992 (gi|197093531); YP_001217666 (gi|147675484); CAL12564 (gi|122089712); BAD14977 (gi|46093563); Or the flagellin of CAD05707 (gi|16503200), all to quote mode, by it, the content whole when the application's priority date is incorporated to herein.

C. microorganism lysate

Embodiment of the present invention also comprise pharmaceutically acceptable compositions, and it comprises the lysate without pathogenic microbes, antiinflammatory and one or more pharmaceutical excipients substantially, and wherein said compositions is aseptic and does not basically contain pathogenic microbes.Microorganism lysate is normally through supersound process; Homogenate; Irradiate; By air pressure, breathing, detergent or enzyme method and combination cracking thereof.A particular aspects, before cracking, in process or afterwards, irradiate microorganism lysate with UV.Microorganism lysate can include but not limited to antibacterial, fungus or viral lysate.In certain embodiments, microorganism lysate is antibacterial lysate.Therefrom prepare the not necessarily poisonous microorganism of microorganism of lysate, and can not be poisonous microorganism conventionally.Aspect of the present invention comprises from the lysate to the limited antibacterial of experimenter's health effect.Affect limited the finger tissue to experimenter, organ or system at least, at the most or about 1,2,3,4,5,6,7,8,9 or 10 day time and produce extremely low side effect and inapparent damage.

Compositions of the present invention does not need directly from the poisonous biology that will protect or treat for it.Antibacterial can be from haemophilus (Haemophilus), but is not limited to haemophilus.Can identify and in experimenter, form the antibacterial that minimal side effect threatens.In some aspects, antibacterial is hemophilus influenza, particularly can not typing hemophilus influenza (NTHi) (Clement etc., 2008; Clement etc., 2009; Evans etc., 2010; Tuvim etc., 2009).

The protein concentration of microorganism lysate can be at least about, approximately or at the most approximately 0.5,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or 10mg/ml, comprise all values and scope therebetween.In some aspects, the protein concentration of microorganism lysate be at least about, about 10mg/ml approximately or at the most.

Embodiment of the present invention comprise the microorganism lysate that can use by respiratory tract.In some aspects, use by suction.On the other hand, compositions is form atomization or that can be sucked by experimenter.In certain embodiments, lysate compositions comprises antiinflammatory, comprises steroid and on-steroidal AID (NSAID).For other details, consult U.S. Patent application 11/830,622 " Compositions and methods for stimulation of lung innate immunity " Dickey etc., be incorporated to herein to quote mode entirety.

B. host or self component

From experimenter or host's cell with the generation that many molecules of tissue can stimulate, enhancing or Promote immunity are replied.These parts are called host or self part or component, comprise the micromolecule discharging from injured, that coerced or dying cell; Participate in the component that microorganism kills and wounds or neutralizes; Cytokine; With the macromole discharging from cell or tissue.

1. micromolecule host compound

To injured, cell that coerced or dying is relevant or the micromolecule that therefrom discharges as ATP (ATP), uric acid (urate) and adenosine.The approach of the receptor of many these molecules and adjusting inflammation thereof is fully definite.Inflammation is immune system for infecting or stimulating first one of replying of making.The chemokines that inflammation is discharged by injured cell stimulates, and acts on the physical barriers of setting up for infecting diffusion, and is removing the healing that promotes any damaged tissue after pathogen.The chemokines (histamine, Kallidin I, serotonin, leukotriene also have prostaglandin) producing in inflammatory process makes pain receptor sensitivity, causes affected part vasodilation, and attracts phagocyte, particularly neutrophil cell.Then neutrophil cell convenes other leukocyte and the lymphocytic factor to trigger immune other parts by release.

The micromolecule host component that can be included in StIR compositions of the present invention comprises ATP, adenosine, histamine, Kallidin I, serotonin, leukotriene, prostaglandin.

2. the outer host's part of born of the same parents

Instruct microorganism to kill and wound and/or signal transduction in outside the born of the same parents of working host protein as complement, pentraxins, alexin and cathelicidin.The frequent composing type of these molecules exists, but until they are by being combined with microbial product or being cut by Proteolytic enzyme or some other activation mechanisms just carry out signal transduction after activating.In addition, its generation can improve.In some aspects, these protein are in activation form (Activation In Vitro or processing, or pass through engineered protein).

Complement system is the biochemistry cascade that pathogen is removed in help from organism.Complement system is non-habitual and the larger immune part that do not change in life at individuality; Therefore it belongs to innate immune system.But it can be raised and be played a role by adaptive immune system.

Complement system is made up of many small protein matter of finding in blood, usually used as the form circulation of non-activity proenzyme.In the time stimulated by one of several triggering agent, the protease cutting specified protein in this system, with the release cells factor the further amplification cascade of cutting of startup.The final result of this activation cascade is the extensive amplification of replying and the activation of cell killing membrane attack complex.Exceed 20 kinds of protein and protein fragments and formed complement system, comprise serum proteins, serous coat protein and cell-membrane receptor.These protein are mainly synthetic in liver, and they account for approximately 5% of serum globulin fraction.

The complement system component that can be included in StIR compositions includes but not limited to C1-complex (C1q, C1r, C1s and C1qr2s2), C1r2s2, C4, C2, C4a, C4b, C2a, C2b C3-invertase (C4b2a complex), C3a, C3b; C5 convertase (C4bC2aC3b complex), decay accelerating factor (DAF), factor B, C3bB, factor D, Ba, Bb, C3bBb, C3bBbC3b, C5, C5a, C5b, C6, C7, C8, C9, and membrane attack complex (membrane attack complex, MAC) (C5b6789).

Pentraxins is the protein families conventionally with Ca-dependent ligand binding and the flat β-jellyroll construction of characteristic (with the similar of legume lectin element)." short " pentraxins comprises serum amyloid P component (SAP) and c reactive protein (CRP)." length " pentraxins comprises PTX3 (molecule that cytokine regulates) and several neural pentraxins.

Alexin is the little cationic protein that is rich in cysteine all existing in vertebrates and invertebrates.They have activity to antibacterial, fungus and much tunicate and acapsular virus.They are made up of 18-45 aminoacid, comprise that 6 (in vertebratess) are to 8 conservative cysteine residues.Immune cell contains these peptides, to help to kill the antibacterial being engulfed in for example neutrophil cell and nearly all epithelial cell.Most of alexins are by being combined and playing a role with microbial cell film, once and embed, just forming hole sample film damaged, it allows essential ion and nutrient to flow out.

The alexin that can be included in StIR compositions of the present invention includes but not limited to α-alexin (DEFA1, DEFA1A3, DEFA3 and/or DEFA4), beta-alexin (DEFB1, DEFB4, DEFB103A/DEFB103B to DEFB107A/DEFB107B, DEFB110 to DEFB133) and/or θ-alexin (DEFT1P).

Cathelicidin antimicrobial peptide is the peptide family being present in the lysosome of polymorphonuclear leukocyte (PMN).The member of Cathelicidin family antimicrobial polypeptide is characterised in that to have the region (cathelin domain) of high conservative and the cathelicidin peptide domain of alterable height.From many different mammalian species, separate cathelicidin peptide.Cathelicidin finds at first, but from many other cells (comprising by antibacterial, virus, fungus or hormone 1 macrophage and epithelial cell that 25-D activates), has also found cathelicidin since then in neutrophil cell.The cystatin of Cathelicidin family and histone enzyme family has primary sequence homology, thinks in this protease suppresses, to have the amino acid residue of importance although generally lack.

3. cytokine

Cytokine is a class signaling molecule that is widely used in intercellular communication.They are protein, peptide or glycoprotein.Term cytokine comprises large and various polypeptides for modulating Zijia family, and its cell by the different embryo's origins of whole body extensively produces.The effect of cytokine can be autocrine, paracrine and endocrine.Cytokine is to congenital most important with generation and function adaptive immune response, although be not limited to only immune system.They are often secreted by the immunocyte that runs into pathogen, activate thus and raise other immunocytes, the replying this pathogen with enhancing system.

The antimicrobial defence of cytokine stimulating innate immunity cell (particularly epithelial cell), as IL-17, IL-22, IFN-y.In some cases, this has represented the amplification of adaptability innate immune system to congenital inflammation, while producing lL-17 as Th17 cell.In other cases, cytokine is by discharging as the cell of an adaptive immune system part, for example epithelial cell, mesenchymal cell or dendritic cell.

The cytokine that can be included in StIR compositions of the present invention comprises IL-1 superfamily 1 ((IL-1Ra), IL-18, IL-33); IL-6 sample/gp130 utilizes family (IL-6, IL-11, IL-27, IL-30, IL-31, oncostatin M, leukaemia inhibitory factor, ciliary neurotrophic factor, Cardiotrophin1); IL-10 family (IL-10, IL-19, IL-20, IL-22, IL-24, IL-26); Interferon type III (IL-28, IL-29); Common γ-chain family (IL-2/15, IL-3, IL-4, IL-7, IL-9, IL-13, IL-21); IL-12 family (IL-12, IL-23, IL-27, IL-35), IL-5; IL-8; IL-14; IL-16; IL-17/25; IL-32; CCL chemotactic factor (CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28); CXCL chemotactic factor (CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17); CX3CL-1; XCL1; XCL2; TNF (part) superfamily (4-1BB part, the B cell-stimulating factor, FAS part, lymphocytotoxin, OX40L, RANKL, TRAIL); Noble cells is because of submanifold (CD70, CD153, CD154); Interferon (IFN-I α (2a of PEGization, the 2b of PEGization), IFN-I β (1a, 1b)), IFN-II γ and IFN-III.

4. macromole host part

Macromole or its fragment can discharge from extracellular matrix, cell surface or cell interior, and activate the signal transduction of innate immunity, as dectin, versican (versican), HMGB-I, DNA and RNA.Conventionally, these macromole are hidden under normal circumstances and do not contact with target receptor, this be hidden in cell interior or by molecule or intermolecular interaction shelter.After cell rupture or the cell surface protein of substrate hydrolysis and demonstrating after signal section (or some similar mechanism), they are released and target acceptor interaction.

II. polypeptide and peptide combinations

In certain embodiments, the present invention relates at least one polypeptide or peptide (for example, polypeptide section) or derivatives thereof or variant.As used herein, " protein ", " polypeptide ", " peptide ", " polypeptide or peptide combinations " or " polypeptide or peptide compounds " refer generally to protein or the polypeptide of (but being not limited to) at least 5 aminoacid or amino acid analogue (whole amino molecules, see below).Above-described all " polypeptide or peptide " terms are used interchangeably in this article.

In certain embodiments, the large I of described at least one polypeptide or peptide molecule includes but not limited to such molecule, it has at least, at the most or approximately 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, 500, 1000 to approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 100, 500 or more amino acid residue, and wherein derivable any value or scope.The present invention includes the continuous amino acid of any sequence or those length of its analog of discussing herein.

The section of polypeptide or peptide or fragment comprise 1,2,3,4,5,6,7,8,9,10,15,20,30,40,50,60,70,80,90,100,150,200,300,350,400,450 amino acids to 10,15,20,30,40,50,60,70,80,90,100,150,200,300,350,400,450,500,550,600 amino acids (comprising all values and scope therebetween) of disclosed herein or sequence that mention.

As used herein, " amino molecule " refers to any aminoacid known to persons of ordinary skill in the art, amino acid derivativges or amino acid analog thing.In certain embodiments, the residue of polypeptide or peptide molecule is continuous, interrupts the sequence of amino molecule residue without any non-amino molecule.In other embodiments, sequence can comprise one or more non-amino molecular moieties.In certain embodiments, the residue sequence of polypeptide or peptide molecule can be interrupted by one or more non-amino molecular moieties.

Therefore, term " polypeptide or peptide combinations " comprises so amino molecular sequences, and it comprises at least one in 20 kinds of common amino acids in natural synthetic protein, or aminoacid at least one modification or rare.

In certain embodiments, polypeptide or peptide combinations comprise at least one protein, polypeptide or peptide.Relating in the method for TLR agonist compositions, polypeptide or peptide can have all or part of aminoacid sequence of flagellin polypeptide (as SEQ ID NO:2 or homeopeptide).In certain embodiments, the compositions that contains protein, polypeptide or peptide is generally not basically contain separately toxin, pathogen and harmful immunogenic protein or peptide or synthetic protein or peptide.In some aspects, polypeptide is restructuring or synthetic aminoacid sequence.

Polypeptide or peptide combinations can be prepared by any technology well known by persons skilled in the art, comprise by standard molecular biological technique marking protein, polypeptide or peptide, and from natural origin isolated polypeptide or peptide, or chemically synthesized polypeptide or fret peptide.Can use technology disclosed herein or known to persons of ordinary skill in the art amplification and/or express the coding region of these polypeptide or peptide.Or the multiple commercialization preparation of protein, polypeptide and peptide is known to those skilled in the art.

In certain embodiments, polypeptide or peptide combinations can be purification.Usually, " purification " refers to remove through fractionated specific protein, polypeptide or the peptide combinations of multiple other protein, polypeptide, peptide and other molecules and compound, and described compositions has retained its activity substantially, this can assess for protein determination specific or protein, polypeptide or peptide that expect by for example known to persons of ordinary skill in the art.

Substantially the component that contains any protein, polypeptide or peptide all can be considered for compositions disclosed herein and method.Consider in certain embodiments, compositions aerosol formulation or spraying or atomization or sprayable can allow compositions more accurately or to be more easily applied to respiratory system by suction, breathing etc.

A. polypeptide or peptide variant and derivant

The aminoacid sequence variant of protein of the present invention, polypeptide and peptide or derivant can be to replace, insert or disappearance variant, and comprise amino acid analogue or derivant.It is not vital one or more residue to function or immunogenicity activity that disappearance variant lacks in native protein.The disappearance variant of another common type is to lack secretory signal sequence or instruct the signal sequence of protein bound to the specific part of cell, or cross-film district or unwanted other functional sequences of activity in vivo.Insertion mutation body is usually included in substance on the non-end site of polypeptide.This can comprise insertion immunocompetence epi-position or only insert single residue.End interpolation is discussed hereinafter, has been called fusion rotein.

Replace variant and conventionally on one or more sites of polypeptide or peptide, contain an aminoacid and another amino acid whose replacement, and can design to regulate one or more characteristics, as the stability to Proteolytic enzyme cutting, but do not lose other functions or characteristic.Such replacement is preferably guarded, and being about to an amino acid substitution is the aminoacid with similar shape and electric charge.Conservative replace with known in the artly, and comprise for example following change: alanine becomes serine; Arginine becomes lysine; Agedoite becomes glutamine or histidine; Aspartic acid becomes glutamic acid; Cysteine becomes serine; Glutamine becomes agedoite; Glutamic acid becomes aspartic acid; Glycine becomes proline; Histidine becomes agedoite or glutamine; Isoleucine becomes leucine or valine; Leucine becomes valine or isoleucine; Lysine becomes arginine; Methionine becomes leucine or isoleucine; Phenylalanine becomes tyrosine, leucine or methionine; Serine becomes threonine; Threonine becomes serine; Tryptophan becomes tyrosine; Tyrosine becomes tryptophan or phenylalanine; Valine becomes isoleucine or leucine.

Term " biological function equivalent " is known in the art, and has carried out in this article further specific definition.Therefore, biological function equivalent can have such sequence, wherein approximately 70,75,80,85,90,95,96,97,98, the identical or functional equivalent of the aminoacid of 99% aminoacid and polypeptide or peptide or its variant or analog or derivant, and provide similar biological activity/replying flagellin or other TLR agonist.

It is below the discussion that the aminoacid based on changing polypeptide or peptide produces (or the even improving) second filial generation molecule being equal to.For example, other aminoacid of the replaceable one-tenth of some aminoacid in polypeptide or peptide, and not obvious forfeiture given activity, as the enhancing of immunne response.Because what conventionally limit protein function activity is interaction ability and the characteristic of polypeptide or peptide, thus can polypeptide or peptide sequence with and corresponding DNA encoding sequence in carry out some amino acid substitution, but produce the protein with similar characteristic.Therefore, the inventor considers to carry out multiple change in the DNA sequence of coding polypeptide of the present invention or peptide, but its Purificatiou of not obvious forfeiture or activity, as discussed below.

In the time producing these and change, hydrophilic index that can considered amino acid.Hydrophile amino acid index is generally this area and understands (Kyte & Doolittle, 1982) giving importance in protein interaction biological function.Be recognized that, amino acid whose relatively hydrophilic characteristics determined the secondary structure of gained protein, it defines the interaction of (such as enzyme, substrate, receptor, DNA, antibody, antigen, immunocyte and systems etc.) such as protein and other molecules, cell, tissues then.

In this area, also it should be understood that and can on hydrophilic basis, effectively carry out similar amino acid whose replacement.Be incorporated to United States Patent (USP) herein 4,554,101 descriptions to quote mode, the highest local average hydrophilic (determining by closing on amino acid whose hydrophilic) of protein is relevant to the biological characteristics of protein.As United States Patent (USP) 4,554, describe in detail in 101, for amino acid residue is given following hydrophilicity value: arginine (+3.0); Lysine (+3.0); Aspartic acid (+3.0 ± 1); Glutamic acid (+3.0 ± 1); Serine (+0.3); Agedoite (+0.2); Glutamine (+0.2); Glycine (0); Threonine (0.4); Proline (0.5 ± 1); Alanine (0.5); Histidine ^*-0.5); Cysteine (1.0); Methionine (1.3); Valine (1.5); Leucine (1.8); Isoleucine (1.8); Tyrosine (2.3); Phenylalanine (2.5); Tryptophan (3.4).

Also it should be understood that the replaceable one-tenth of aminoacid has another aminoacid of similar hydrophilicity value, but still produce the protein that be equal to biology and immunology is equal to.In this change, the preferably amino acid substitution of its hydrophilicity value in ± 2, particularly preferably those in ± 1 of its hydrophilicity value, even more preferably its hydrophilicity value in ± 0.5 those.

As noted above, amino acid substitution is generally for example, based on the substituent relative similarity of amino acid side chain, their hydrophobicity, hydrophilic, electric charge, size etc.The exemplary replacement that multiple above-mentioned feature is taken into account is well known to those skilled in the art, and comprising: arginine and lysine; Glutamic acid and aspartic acid; Serine and threonine; Glutamine and agedoite; And valine, leucine and isoleucine.

Also can modify internal amino acid and/or aminoterminal and/or the c-terminus of polypeptide of the present invention or peptide compounds, to produce other compounds of the present invention, i.e. polypeptide or peptide derivant.Aminoterminal is modified and is comprised (for example ,-NHCH that methylates ₃or-N (CH ₃) ₂), acetylation (for example, with acetic acid or its halo derivatives, as α-monoxone, alpha bromoisobutyric acid or alpha-iodine acetic acid), add benzyloxycarbonyl group (Cbz), or with contain with RCOO-define carboxylic acid functional or R-SO ₂any sealing base sealing aminoterminal of the sulphonyl functional group of-definition and similar group, wherein R is selected from alkyl, aryl, heteroaryl, alkylaryl etc.Also can mix without amino acid (thereby do not have N end amino) at N end, to reduce the sensitivity to protease or to limit the conformation of polypeptide or peptide compounds.

C-terminus is modified and is comprised with carbonylamino group replacement free acid or form annular lactams at c-terminus, to introduce structural limitations.Also can make peptide cyclisation of the present invention, or mix without amino or without the residue of carboxyl at the end of peptide, thereby there is no terminal amino group or carboxyl, reduce sensitivity to protease or the conformation of restriction peptide.The C end functional group of the compounds of this invention comprises amide, amide low alkyl group, amide two (low alkyl group), lower alkoxy, hydroxyl and carboxyl, and lower member ester derivant and officinal salt.

The natural side chain of 20 kinds of genetic coding aminoacid (or stereoisomer D aminoacid) can be replaced with to other side chains, for example, use such as the group of alkyl, low alkyl group, 4,5,6 to 7 Yuans alkyl of ring-type, amide, amide low alkyl group, amide two (low alkyl group), lower alkoxy, hydroxyl, carboxyl and lower member ester derivant thereof and with 4,5,6 to 7 element heterocycles.Particularly, can use proline analogs, wherein proline residue ring size becomes 4,6 or 7 members from 5 Yuans.Cyclic group can be saturated or unsaturated, if undersaturated can be aromatics or non-aromatic.Heterocyclic group preferably contains one or more nitrogen, oxygen and/or sulfur heteroatom.The example of this type of group comprises furan a word used for translation base (furazanyl), furyl, imidazolidinyl, imidazole radicals, imidazolinyl, isothiazolyl, different

azoles base, morpholinyl (for example morpholino),

azoles base, piperazinyl (for example 1-piperazinyl), piperidyl (for example piperidino, piperidines generation (piperidino)), pyranose, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridine radicals, pyrimidine radicals, pyrrolidinyl (for example 1-pyrrolidinyl), pyrrolinyl, pyrrole radicals, thiadiazolyl group, thiazolyl, thienyl, thio-morpholinyl (for example thiomorpholine generation (thiomorpholino)) and triazolyl.These heterocyclic groups can be that replace or unsubstituted.Group be replace time, substituent group can be alkyl, alkoxyl, halogen, oxygen, or replacement or unsubstituted phenyl.

Also can such as, by phosphorylation and additive method (described in Hruby etc. (1990)) modified polypeptide or peptide expediently.

Peptide compounds of the present invention is also used for having similar bioactive non-peptide compound as construction module.Those skilled in the art is recognized that, useful multiple technologies build such compound, it has identical or similar expectation biological activity with leader peptide compound, but (consult at dissolubility, stability with to thering is more favourable activity than leader peptide compound aspect the sensitivity of hydrolysis and proteolysis, Morgan and Gainor, 1989).These technology comprise peptide backbone are replaced with to the skeleton being made up of phosphate ester, amidate, carbamate, sulfonamide, secondary amine and N-methylamino acid.

In addition, compound of the present invention can contain one or more intramolecular disulfide bonds.In one embodiment, peptide monomer or dimer comprise at least one intramolecular disulfide bond.In some preferred embodiments, peptide dimer comprises two intramolecular disulfide bonds.Can be by making the cysteine residues oxidation in peptide core sequence form this disulfide bond.In one embodiment, implement by oxidant type and the concentration of selecting effectively to optimize the isomer formation of expecting the control that cysteine key forms.For example, in the time that oxidant is DMSO, preferentially complete the oxidation of (compared with forming intermolecular disulfide bond) peptide dimer, to form two intramolecular disulfide bonds (having on every peptide chain).In certain embodiments, control the formation of cysteine key with sulfhydryl protected base by selectivity in peptide building-up process.

Other embodiments of the present invention provide the analog of these disulfide derivatives, and one of them sulfur is by CH ₂other isosteres of group or sulfur are replaced.Can use methods known in the art, by in molecule or intermolecular displacement, prepare these analog by the compounds of this invention, wherein each core sequence contains at least one Cys (C) or homocysteine residue and replaces the alpha-amido-γ-butyric acid of second C residue (for example to consult, Barker etc., 1992 and Or etc., 1991).Those skilled in the art can easily understand, and also can use other congeners of alpha-amido-γ-butyric acid and homocysteine to carry out this displacement.

Except above-mentioned cyclisation strategy, also can use other non-disulphide peptide cyclisation strategies.This type of alternative cyclisation strategy comprises for example amide cyclisation strategy and relates to those strategies that thioether bond forms.Therefore, compound of the present invention can have the cyclisation form existence of thioether bond in molecule lactam bond or molecule.For example, can synthetic peptide, wherein core sequence cysteine is replaced by lysine, and second cysteine replaced by glutamic acid.Then, can form cyclic monomer by the amido link between the side chain of these two residues.Or, can synthetic peptide, wherein core sequence cysteine is replaced by lysine.Then be connected to form cyclic monomer by the thioether between the side chain of lysine residue and second cysteine residues of core sequence.Like this, except disulphide cyclisation strategy, also can easily use amide cyclisation strategy and thioether cyclisation strategy, carry out cyclisation compound of the present invention.Or the acetic acid of available alpha-substituted adds cap to the aminoterminal of peptide, wherein alpha-substituted base is leaving group, as alpha-halogen acetic acid, for example, α-monoxone, alpha bromoisobutyric acid or alpha-iodine acetic acid.

The U.S. Patent Application Serial Number 10/844,933 of submitting to 12 days Mays in 2004 that below description comprises is incorporated to herein to quote mode entirety.Water-soluble polymer has polypeptide or the peptide for the treatment of importance as Polyethylene Glycol (PEG) can be used for covalent modification.Think adhering to of this base polymer to have strengthened biological activity, improved water solublity, and strengthen the resistance to protease digestion.For example, be reported that PEG is covalently attached to therapeutical peptide, as interleukin (Knauf, etc., 1988; 15064; Tsutsumi etc., 1995), interferon (Kita etc., 1990), catalase (Abuchowski etc., 1977, superoxide dismutase (Beauchamp etc., 1983, and ADA Adenosine deaminase (Chen etc., 1981) on, extend their half-life in vivo, and/or reduced their immunogenicity and antigenicity.

Compound of the present invention also can comprise one or more water-soluble polymer parts.Preferably, these polymer are covalently attached on this compound.Water-soluble polymer can be the copolymer, carboxymethyl cellulose, glucosan, polyvinyl alcohol, polyvinylpyrrolidone of for example Polyethylene Glycol (PEG), ethylene glycol/propylene glycol, poly--1,3-dioxolane, poly--1,3,6-tri-

alkane, ethylene/copolymer-maleic anhydride, polyamino acid (homopolymer or randomcopolymer), poly-(n-ethylene pyrrolidone) Polyethylene Glycol, propylene glycol homopolymer, polyoxypropylene/oxygen ethylene copolymer and polyoxyethylene polyhydric alcohol.

Can use any method in number of chemical method that compound of the present invention is attached to (for example, PEG) on water miscible polymer, for example, water-soluble polymer is connected on the receptor binding moiety of molecule (, peptide+spacer).Typical embodiment is used the single contact that adheres to, for water-soluble polymer is covalently attached to receptor binding moiety, but in some alternative embodiment, can use multiple contacts that adhere to, comprise other variations, wherein different types of water-soluble polymer is attached to receptor binding moiety in different adhering on contact, and it can comprise that covalent attachment joins on spacer and/or one or two peptide chains.

PEG reagent includes but not limited to mPEG2-NHS, mPEG2-ALD, multi-arm PEG, mPEG (MAL) ₂, mPEG2 (MAL), mPEG-NH ₂, mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEG-dibasic acid esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET, exclusive-OR function PEG (NH ₂-PEG-COOH, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-MAL), PEG acrylate (ACRL-PEG-NHS), PEG-phospholipid (for example mPEG-DSPE), the multi-arm PEG of SUNBRITE series, comprise the GL series of the PEG based on glycine activating by the selected chemical method of those skilled in the art, the PEG that any SUNBRITE activates (includes but not limited to carboxyl-PEG, p-NP-PEG, Tresyl-PEG, aldehyde PEG, acetal-PEG, amino-PEG, sulfydryl-PEG, maleimide-PEG, hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-aldehyde, carboxylic acid anhydrides type-PEG, the PEG-phospholipid of functionalization, with those skilled in the art according to its application-specific and use select other similar and/or suitable active PEG).

The quantity of the polymer molecule adhering to can change; For example, can by 1,2,3 or more polymer be attached on polypeptide of the present invention or peptide.Multiple polymer that adhere to can be identical or different chemical parts (for example, the PEG of different molecular weight).In some cases, in attachment reaction, in the ratio of polymer molecule and peptide molecule and reactant mixture, the total concentration of each can affect the degree that polymer adheres to (be attached to the quantity of the polymer moieties on peptide and/or be attached with the sum of the peptide of polymer).Generally speaking, the optimal proportion of polymer and peptide is (with regard to reaction efficiency, excessive unreacted peptide and/or polymer moieties is not provided) will determine based on following factor: the expected degree adhering to as polymer is (for example, single, two, third-class), the molecular weight of selected polymer, polymer be branch or unbranched, and the reaction condition of concrete adherence method.

Aspect other, can derive the compounds of this invention by adding insoluble polymer.Representative insoluble polymer includes but not limited to polyphosphazene (polyphosphazine), polyvinyl alcohol, polyamide, Merlon, poly-alkylene (polyalkylene), polyacrylamide, poly-alkyl diol, polyethylene glycol oxide, polyalkylene terephthalates (polyalkylene terephthalate), polyvinylether, polyvinyl ester, polyethylene halides, polyvinylpyrrolidone, polyglycolide, polysiloxanes, polyurethanes, polymethyl methacrylate, polyethyl methacrylate, polybutyl methacrylate, polyisobutyl methacrylate, the own ester of polymethylacrylic acid, polymethylacrylic acid isodecyl ester, polylauryl methacrylate, polymethyl acid phenenyl ester, polymethyl acrylate, polyacrylic acid isopropyl ester, polyisobutyl acrylate, polyoctodecyl acrylate, polyethylene, polypropylene, Polyethylene Glycol, polyethylene glycol oxide, polyethylene terephthalate, polyvinyl acetate, polrvinyl chloride, polystyrene, polyvinylpyrrolidone, pluronic and polyvinyl phenol and copolymer thereof.

The natural polymer that is used for the synthetic modification of derivant of the present invention includes but not limited to alkylcellulose, hydroxy alkyl cellulose, cellulose ether, cellulose esters and celluloid.The member of the synthetic modification natural polymer of wide range of types includes but not limited to methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl emthylcellulose, hydroxy butyl methyl cellulose, cellulose acetate, cellulose propionate, acetylbutyrylcellulose, CAP, carboxymethyl cellulose, Triafol T, cellulose sulfate sodium salt and acrylic acid and methacrylate and alignic copolymer.

In some aspects, can modify or derivative polypeptide of the present invention or peptide by the sugar of interpolation glycosyl or modification.The invention provides polypeptide and peptide derivant, the ribotide of its sugar that contains modification, modification and the sugared conjugate of modification.In the sugar compounds of modification of the present invention, sugar moieties is preferably sugar, deoxysaccharide, amino sugar or N-acyl group sugar.Term " sugar " and equivalent " glycosyl " thereof refer to monomer, dimer, oligomer and polymer.Also can utilize modification group to make sugar moieties functionalization.Modification group conventionally by with sugar moieties on amine, sulfydryl or hydroxyl (for example primary hydroxyl) put together and be conjugated on sugar moieties.In one embodiment, modification group adheres to by the amine moiety on sugar (for example, by amide, urethanes or carbamide (it forms with the reacting of reactive derivative of modification group by amine)).

Any sugar all can be used as the sugar of conjugate of the present invention.This type of sugar includes but not limited to glucose, galactose, mannose, trehalose and sialic acid.Other useful sugar comprise amino sugar, as glycosamine, galactosamine, mannosamine, sialic 5-amine analog etc.Sugar can be the structure that occurring in nature is found, or the site that can be modified to be provided for to put together extra modification group.

It will be understood by those skilled in the art that described structure and compositions can be applied in general manner different types of glycosyl, modify the modification glycosyl of glycosyl, activation and modify in glycosyl conjugate.

III. the stimulation of lung defence

The inventor has used the model of mice as lung infected by microbes.In some research, untreated mice has 100% mortality rate, but treated mice is subject to highly protection.Do not wish to be subject to any specific mechanism or theoretical constraint, believe that protection is the activation due to local defense or innate immunity.After measured experimenter's single and the effect that repeats to contact the present composition, do not observe significantly serious pathology, as too early death, lose weight or behavior change.

A non-limiting benefit of the present invention is that it can be sent fast and expediently and tell on.Equally, described compositions can produce and be easy to preserve economically in a large number, and can be easily transported to outside hospital environment by people.Conventionally, using with method of the present invention of the present composition causes even at least certain the killing and wounding or suppress that have to intrusion pathogen before cell enters.Kill and wound outward and enter cell or (preventative) uses compositions before (property in advance) rather than pathogen contact after pathogen contact in the situation that by escaping born of the same parents in some pathogen, consider to kill and wound in born of the same parents that described compositions and correlation technique promote the local-acknowledgement because strengthening in lung or amplifying to cause.Consider that described compositions and correlation technique have for various respiratory road pathogen or produce protectiveness or therapeutic is replied.

StIR compositions gives individual protection or treatment can, because the extensive activity of respiratory tract antimicrobial mechanism extends to extra microbial pathogens classification, comprise gram negative bacteria, intracellular bacteria, fungus and virus.The reagent of describing in the application can be simplified storage and the deployment of counter measure.Equally, the compositions and methods of the invention can be got rid of the difficulty of Rapid identification special pathogen in bioweapons attack or other contacts or potential contact event procedure.

In addition it is significant, producing and buy the economic benefit in multiple civilian and biophylaxis situation with the reagent of application.For example strengthen local epithelium mechanism, often to suffer from neutrophil cell especially attractive in reducing disease or the impaired experimenter of adaptive immunity function (experimenter that immunity reduces).The method conventionally in part but not whole body work, and provide extensive effect for multiple pathogens.This effect is fast, and attractive in biophylaxis, medical science and epidemic diseases situation.

Do not have adaptive immunity vaccine can with influenza or urgent respiratory virus epidemiological process in strengthen normal host in the congenital defence capability of lung be valuable.Have and appear suddenly or the antibacterial outburst of drug resistance biology may be also such situation, it is useful wherein strengthening congenital lung defence.Similarly, in epidemic diseases process, can be beneficial to look after patient to medical personnel's protection, restriction diffusion.

Many people in community reduce for a long time for the defence of infecting, as suffer from the patient of diabetes and due to autoimmune disease or prevent that transplant rejection from taking the patient of immunosuppressive drug.These people may especially benefit from the enhancing that lung is defendd in epidemiological process or in the probability rising of contact microorganism.Even more surprising, carry out chemotherapeutical cancer patient (it has temporary transient but serious immune defence and reduces) and can benefit from temporary protection.Pneumonia often occurs these patients, and is infectious dead primary factor.Many chemotherapeutic agents (as alkylating agent and nucleoside analog) cause that serious temporary neutrophil cell reduces disease.At first, neutropenia patient susceptible is in the bacterial pneumonia being caused by visible biology in from normal host and hypotoxic antibacterial (as having a liking for maltose Stenotrophomonas (Stenotrophomonas maltophilia)).Neutrophil cell reduces disease overtime, and patient starts susceptible in the infection of low virulence fungus (especially aspergillus species).

Can stimulate the defence of lung, to provide temporary protection in the extended period of neutrophil cell minimizing disease.Other cancer patients' (as accepted those patients of fludarabine or antilymphocyte antibody, or accepting those patients of neurocalcin inhibitor and steroid after hematopoietic stem cell transplantation) adaptive immunity sustains damage.The intermittent type that these patients also can benefit from lung immunity stimulates, and to provide protection for the invasion and attack of the air flue fungus and bacterium of growing decided at the higher level but not officially announced, or provides protection for the invasion and attack of epidemic virus.Community's property outburst of seasonal respiratory tract " flu " virus (as parainfluenza virus and RSV) can cause lethal pneumonia in the patient of these irresistances, and can from nasal mucus, identify rapidly the infection of many these viruses.

After infection, the identification of microorganism mainly mediates (Medzhitov and Janeway, 1997) by the one group of germline coding molecule that is called pattern recognition receptors (PRR) on innate immunity cell.These expression of pattern recognition receptors are the protein of membrane-bound or solubility, and it identifies constant molecular structure, are called the molecular pattern that pathogen is relevant (PAMP) (Janeway and Medzhitov, 2002).The molecular pattern that pathogen is relevant is unique, conservative, and necessary microbial components (as LPS) is structurally different from host's molecule (Medzhitov and Janeway, 1997; Janeway and Medzhitov, 2002).

Most of multicellular organisms have " innate immune system ", and it does not change in life at biology.On the contrary, adaptive immunity is the replying pathogen that changes in life and occur at individuality.The biology with adaptive immunity also has innate immunity, and because the many mechanism between system are common, therefore be not always can be participating in sketching out between each component of each immunity clear and boundary fast, although have obvious difference on operating.High vertebrates and all mammals all have innate immune system and adaptive immune system simultaneously.

A. innate immune system

Adaptive immune system can be after primary infection several days or a few week have effect.But, under the lasting attack of most of biologies in pathogen, must suppress it by the innate immune system of faster response.Innate immunity is defendd pathogen by the rapid answer of " congenital " mechanism coordination, and described " congenital " mechanism is identified conservative pathogen component widely.Great majority research to innate immunity is conceived to leukocyte, as neutrophil cell, macrophage and natural killer cell.But epithelial cell plays a crucial role in congenital defence, it the mechanical barrier that provides microorganism to enter is provided, carries out signal transduction to leukocyte, and direct pathogen kill.Importantly, all these defence can respond to the perception to microorganism and host products and highly be induced.In healthy lung, innate immunity epithelium function is that baseline is low-level.This is killed and wounded with release of cytokines and is represented by low-level spontaneous microorganism, has reflected when the effectively almost low stimulation of persistence in aseptic lower respiratory tract when work of mucociliary clearance mechanism.This is contrary with colon, antibacterial sustainable existence here, and epithelial cell is by sustained activation.Although lung surface is rendered as the large target that microorganism is invaded, near the pulmonary epithelial cells of the activation pathogen of deposition is arranged ideally to carry out microorganism and is killed and wounded.(consulting Evans etc., 2010).Plant and many lower animals do not have adaptive immune system, but depend on its innate immunity.The material in microorganism and non-microorganism source can both be replied by stimulating innate immunity.

Innate immune system has effector lymphocyte and mechanism widely after activation.Have several dissimilar phagocyte, its picked-up also destroys and invades pathogen.Modal phagocyte is neutrophil cell, macrophage and dendritic cell.Another cell type (natural killer cell) is especially good at the cell that break virus infects.Another component of innate immune system is called complement system.Complement protein is the anergy component of blood normally.But when by pathogen or antibody recognition activation, multiple proteins is activated to raise inflammatory cell, wraps up pathogen so that they are more easily engulfed, and form destructive hole on the surface of pathogen.

" First Line " defence comprises the physics and chemistry barrier to infecting, as coated in the mucus of skin and digestive tract and air flue, prevents physically the interaction between host and pathogen.The pathogen that penetrates these barriers meets the antimicrobial molecule (for example, lysozyme) of the constitutive expression of restriction infection." the second line " defence comprises phagocyte (macrophage and neutrophil cell), its (engulfing) foreign substance of can eating.

Phagocytosis relates to chemotactic, wherein by chemotactic chemical substance (as microbial product, complement, damaging cells and leukocyte fragment), phagocyte is attracted in microorganism.After chemotactic, be adhesion, wherein phagocyte is bonded in microorganism.Strengthen adhesion by opsonic action, wherein protein (as opsonin) is coated on bacterium surface.After this, be digestion, wherein phagocyte is stretched out projection, forms the pseudopods of the adventive that eats.Finally, the enzyme in lysosome (comprising reactive oxygen species and protease) digests pathogen.

In addition,, if pathogen has been passed physical barriers, anti-microbial protein can be activated.There are a few class anti-microbial proteins, for example, as acute phase protein (, c reactive protein strengthens and engulfs and activating complement in the time of the C protein binding of itself and streptococcus pneumoniae), lysozyme and complement system.

Complement system is one group of very complicated serum proteins, and it is activated with cascade system.Complement activation relates to three different approach: (a) classical pathway, it identifies antigen-antibody complexes, (b) alternative route, it is spontaneously activation after contacting with pathogenicity cell surface, (c) mannose binding lectin approach, it identifies mannose, and it only occurs conventionally on pathogenicity cell surface.It after complement activation, is the cascade of protein active; This cascade can produce multiple effect, comprises the opsonic action of pathogen, formation and destruction and the inflammation of activation to pathogen of MAC.

Interferon is also anti-microbial protein.These molecules are the protein of the emiocytosis of viral infection.Then these protein are diffused rapidly to adjacent cells, induce these cells to suppress the diffusion of viral infection.In fact, the effect of these anti-microbial proteins is to prevent that viral cell is to cell amplification.

B. adaptive immune system

The further disease that most of mammals that adaptive immune system (also referred to as " acquired immune system ") guarantees to survive after pathogen primary infection generally cause identical pathogen has immunity.The special immunocyte of adaptive immune system based on being called leukocyte (leukocyte), it is produced by the stem cell in bone marrow, and ripe in thymus and/or lymph node.In many species (comprising mammal), adaptive immune system can be divided into: (a) immunity system, its protein that is called immunoglobulin (also referred to as antibody) by the generation of B cell for example, works to antibacterial and virus in body fluid (, blood); (b) cell immune system, it passes through T cell (also referred to as " T lymphocyte "; " T " represents that they occur in thymus) break virus infect cell (also bringing into play other functions).Adaptive immune system is often referred to special pathogen, for example vaccination.

IV. microorganism

Embodiment of the present invention for example comprise, for multiple pathogens or potential pathogen (, the preferential pathogen of NIAID classification A, B and C) carries out compositions and the correlation technique extensively protected.For example, bacterial pneumonia incidence rate with 1/100 people/year in normal host occurs, and is mainly in old man and child, and can be caused by multiple biology.Secondly it be that encapsulated hemophilus influenza causes the most often for streptococcus pneumoniae causes.Other antibacterials as intestinal gram negative bacteria, anaerobe and staphylococcus aureus be the main cause of pneumonia in specific environment (as medical treatment and nursing place).Mycobacterium tuberculosis (Mycobacterium tuberculosis) is hyperinfection, and is the important cause of mortality rate in world wide in history.Carried out most control with antibody in developed country, but multi-drug resistance bacterial strain still continues to cause problem and is divided into classification C biological weapons reagent.Legionella pneumophila is first out identified in the outburst in Philadelphia in 1978, although think that at present it extensively occurs with the low endemicity speed relevant to environment source.Equally, the fungal infection of lung can cause Symptomatic disease in normal host.Histoplasma capsulatum, Blastomyces coccidioides (Coccidiodes immitis), Blastomyces dermatitidis (Blastomyces dermatitidis) and crytococcus neoformans (Crytococcus neoformans) all can cause to part and be exposed to pneumonia relevant under high ambient concentration.The pneumonia being caused by these pathogenic epiphytes is generally self limiting in normal host.Some extra " atypia " microorganisms account for the pith of other pneumonia in normal host as mycoplasma.Think that compositions of the present invention can provide temporary protection fast for the multiple pathogens that causes for example pneumonia or other diseases situation.In some aspects, the present invention can be used for and vaccination scheme combination, thereby provides extra protection for the experimenter who may or contact one or more pathogenicities or potential pathogenic organism.

In particular aspects more of the present invention, the compositions and methods of the invention can be used for prevention or treatment is infected or with contacting of biological weapons/conditionality microorganism or contacting of the infectious agent of experimenter and suction, or reduce its risk.Bacillus anthracis as unique microbial pathogens of terrorist's weapon in the modern times, in the time occurring by respiratory pathways to infect, even if use suitable antibiotic also to there is 75% fatality rate.It is aerobic gram-negative coccobacillus that soil draws hot Frances Salmonella, and it is facultative intra-cellular pathogens.It is hyperinfection, highly pathogenic, and can under severe environmental conditions, survive, and makes it become serious bio-terrorism and threatens, even if it is difficult to propagate (Dennis, 2001) between people.There is vaccine to use, but only have part protectiveness.World Health Organization's estimation, on the urban district that has 5,000,000 residents, the severe toxicity soil of aerosol sprinkling 50kg draws hot Frances Salmonella can cause 250,000 anergy injures and deaths, comprises that 19,000 examples are dead; Center for Disease Control (CDC) (CDC) estimation, the Financial cost of this attack is 5,400,000,000 dollars of every 100,000 exposed population groups (Dennis, 2001).

Other category-A bio-terrorism reagent that can propagate by aerosol are Yersinia pestis, smallpox virus and hemorrhagic fever virus.In addition, can effectively send multiple category-B and C class reagent by respiratory tract.In a word, these biologies comprise in Gram-positive, Gram-negative, cell and extracellular antibacterial, and multiple viral classification.Because may be difficult to first to identify concrete bio-terrorism reagent, local storage for the adaptive immunity vaccine of particular agent and antibiotic complexity and biology (as Bacillus anthracis) even if correct processing still has remarkable toxicity, so stimulate the congenital defence capability of lung to prevent or stop in advance (preempt) or alleviate the infection of the bio-terrorism agent of sending by respiratory tract; This effect can have great public health and be worth.

A. pathogenicity or potential pathogenic microbes

There are many microorganisms to be considered to have pathogenicity, or may have pathogenicity under certain conditions (, conditionality pathogen/microorganism).In some aspects, determine pathogenicity with respect to pulmonary infection.Bacillary microorganism includes but not limited to multiple species that bacillus cereus, yersinia, Franscisella, streptococcus, staphylococcus, pseudomonas, mycobacteria, Burkholderia belong to.Protection to experimenter for concrete bacterial species include but not limited to Bacillus anthracis, Yersinia pestis, soil draws hot Frances Salmonella, streptococcus pneumoniae, staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia cepacia, diphtheria corynebacterium, clostridium (Clostridia spp), shigella (Shigella spp.), Mycobacterium avium (Mycobacterium avium), M.intracellulare, M.kansasii, M.paratuberculosis, M.scrofulaceum, M.simiae, M.habana, M.interjectum, M.xenopi, M.heckeshornense, M.szulgai, M.fortuitum, M.immunogenum, M.chelonae, M.marinum, M.genavense, M.haemophilum, M.celatum, M.conspicuum, M.malmoense, M.ulcerans, M.smegmatis, M.wolinskyi, M.goodii, M.thermoresistible, M.neoaurum, M.vaccae, M.palustre, M.elephantis, M.bohemicam and M.septicum.

B. virus

There are multiple virus and Strain to be considered to pathogenicity or have under given conditions potential pathogenicity.Virus can be divided into one of following seven groups: group I: double-stranded DNA virus; Group II: single-stranded DNA viruses; Group III: diplornavirus; Group IV: sense strand single strand RNA virus; Group V: antisense strand single strand RNA virus; Group VI: reverse transcription diploid single strand RNA virus; Group VII: reverse transcription annular double-stranded DNA virus.Virus comprises Adenoviridae, Arenaviridae (Arenaviridae), Caliciviridae (Caliciviridae), coronaviridae (Coronaviridae), filamentous virus section (Filoviridae), flaviviridae (Flaviviridae), Hepadnaviridae (Hepadnaviridae), herpetoviridae (Herpesviridae) (Alphaherpesviridae (Alphaherpesvirinae), Betaherperesvirinae (Betaherpesvirinae), Gammaherpesvirinae (Gammaherpesvirinae)), Nidovirales, Papillomaviridae (Papillomaviridae), Paramyxoviridae (Paramyxoviridae) (the sick subfamily of secondary mucus (Paramyxovirinae), Pneumovirinae (Pneumovirinae)), Parvoviridae (Parvoviridae) (Parvovirinae (Parvoviridae), Picornaviridae (Picornaviridae)), Poxviridae (Poxviridae) (Chordopoxvirinae (Chordopoxvirinae)), Reoviridae (Reoviridae), Retroviridae (Retroviridae) (Orthoretrovirinae) and/or Togaviridae (Togaviridae).These viruses include but not limited to multiple influenza strain, for example, as bird flu (, H5N1).Protection to experimenter for concrete virus include but not limited to cytomegalovirus (Cytomegalovirus), respiratory syncytial virus etc.

The example of pathogenic virus includes but not limited to influenza A virus, H5N1, Marburg virus, Ebola virus, dengue virus, SARS, yellow fever virus, human respiratory syncytial precursor virus, vaccinia virus etc.

C. fungus

There are many fungal species to be considered to pathogenicity or have under given conditions potential pathogenicity.The protection providing can be for (but being not limited to) Aspergillus fumigatus, Candida albicans, cryptococcus neoformans, histoplasma capsulatum, Blastomyces coccidioides or Pneumocystis carinii and/or Blastomyces dermatitidis.

V. preparation and using

Can use pharmaceutical composition disclosed herein by experimenter's respiratory system.In some aspects, by methods known in the art and device, compositions is deposited in lung.Can in water, prepare StIR compositions, as suitably mixed in hyprolose with surfactant.Also in glycerol, liquid macrogol and its mixture and oil, prepare dispersant.Under common storage and service condition, these preparations contain antiseptic, prevent microbial growth.The medicament forms that is applicable to sucking comprises aseptic aqueous solution or dispersant and the sterile powder for the aseptic inhalation solution of interim preparation or dispersant.In all cases, described form is normally aseptic, and can directly suck or suck by some pilot processs or device.It must be stable under preparation and condition of storage, and must prevent the contamination of microorganism (as antibacterial and fungus).Carrier can be to contain for example solvent or the disperse medium of water, ethanol, polyhydric alcohol (for example, glycerol, propylene glycol and liquid macrogol etc.), its suitable mixture and/or vegetable oil.Can realize by multiple antibacterium and antifungal the prevention of microbial action, such as metagin, chlorobutanol, phenol, sorbic acid, thimerosal etc.

According to treated experimenter's situation with contact or the concrete condition of potential contact, need to necessarily change dosage.Under any circumstance, the people who is responsible for using can determine suitable dosage for individual subjects.In addition, use for people, preparation should meet aseptic, pyrogenicity, general security and the purity rubric of FDA biological product standards place or other similar means requirements.

By active component is mixed in appropriate solvent and prepared aseptic composite with multiple other compositions of enumerating with the amount of needs, while needs, carry out subsequently for example filtration sterilization above.Usually, prepare dispersant by multiple sterile active composition is mixed in sterile carrier, described sterile carrier contains basic dispersant and from other required compositions of enumerating above.In the case of the sterile powder for the preparation of aseptic composite, some preparation methoies are vacuum drying and Freeze Drying Technique, and it produces active component and the powder from any extra required composition of aforementioned aseptic filtration solution.

Can implement (comprising liquid dispenser, metered-dose inhaler (MDI) based on aerosol, aerosol apparatus, dry powder dispersal device etc.) drug delivery of lung/respiratory tract by distinct methods.This type of method and composition is well known to those skilled in the art, and as United States Patent (USP) 6,797,258,6,794,357,6,737,045 and 6,488,953 is indicated, is all incorporated to herein to quote mode.According to the present invention, can be by known in the art for sending at least one pharmaceutical composition by any multiple suction or the nasal devices that suck administering therapeutic agent.Other devices that applicable guiding lung or nose are used are also known in the art.Conventionally, use for carrying out lung, send at least one pharmaceutical composition with the granular size of the downtake that effectively arrives lung or hole.Some instantiations that are suitable for implementing commercially available suction apparatus of the present invention are Turbohaler ^tM(Astra),

(Glaxo),

(Glaxo), Spiros ^tMdevice, AERx that inhaler (Dura), Inhale Therapeutics sell ^tM(Aradigm),

aerosol apparatus (Mallinckrodt),

aerosol apparatus (Marquest Medical Products),

metered-dose inhaler (Glaxo), in powder inhaler (Fisons) etc.

All these type of suction apparatus all can be used for using the pharmaceutical composition in aerosol.This type of aerosol can comprise solution (aqueous solution and non-aqueous solution) or solid particle.Metered-dose inhaler conventionally uses propellant gas and need in breathing process, start.Consult for example WO98/35888; WO94/16970.The respiratory promoter of the mixed powder of Diskus utilization.Consult United States Patent (USP) 5,458,135; 4,668,218; The open WO97/25086 of PCT; WO94/08552; WO94/06498; With European patent application EP 0237507, be incorporated to herein to quote mode entirety.Aerosol apparatus produces aerosol from solution, and the generation granule aerosols such as metered-dose inhaler, Diskus.Include but not limited to nasal spray or nasal drop for the appropriate formulation of using, and can comprise water or the oil solution of StIR compositions.

Can under pressure, produce by nozzle the spraying that comprises pharmaceutical composition of the present invention by the suspension or the solution that force compositions.Can select jet size and configuration, applied pressure and liquid feeding speed, to realize output and the granular size of expectation.Can carry out electron spray, for example, by the electric field being connected with capillary tube or nozzle feed.

Can use pharmaceutical composition of the present invention as jet nebulizer or ultrasonic nebulizer by aerosol apparatus.Conventionally,, in jet nebulizer, compressed air source is for generation of by the high-velocity jets of nozzle.Along with gas is diffused into outside nozzle, produce low-pressure area, it is drawing compositions by connecting the capillary tube of liquid reservoir.While flowing outside effuser from liquid capillaceous, be sheared into unsettled silk and droplet, produce aerosol.Can use configuration, flow velocity and baffle type widely, to realize the performance characteristic of expecting from given blast atomizer.In ultrasonic nebulizer, conventionally use piezoelectric transducer by altofrequency electric energy the mechanical energy for generation of vibratility.This energy delivery, in compositions, produces aerosol.

In metered-dose inhaler (MDI), in tank, contain propellant, compositions and any excipient or other additives and compressed-air actuated mixture.The startup of metering valve discharges mixture as aerosol.

The pharmaceutical composition that utilizes metered-dose inhaler device to use generally comprises fine powder, and it contains compositions of the present invention as the suspending agent in anhydrous medium, for example at the help low suspension of surfactant in propellant.Propellant can be any conventional material for this object, as Chlorofluorocarbons (CFCs), hydrogenated chloride fluorine hydrocarbon, hydrogenated carbon fluorine compounds or Hydrocarbon, comprise Arcton 11, dichlorodifluoromethane, dichloro-tetrafluoro ethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrogenation fluorine alkane-134a), HFA-227 (hydrogenation fluorine alkane-227) etc.

As used herein, " carrier " comprises any and all solvents, disperse medium, supporting agent, coating, diluent, antibacterial and antifungal, isotonic agent and absorption delay agent, buffer, carrier solution, suspension, colloid etc.This type of medium and reagent are known in the art for the purposes of pharmaceutically active substance.Unless any conventional media or reagent are incompatible with active ingredient, otherwise all consider its purposes in described therapeutic composition.Complementarity active ingredient also can mix in described compositions.

Phrase " pharmaceutically acceptable " refers to not produce molecular entity and the compositions of irritated or similar untoward reaction in the time using to experimenter.Containing polypeptide or peptide is known in the art as the preparation of the water composition of active ingredient.

VI. combined therapy

The compositions and methods of the invention can be used in the situation of many treatments or prophylactic applications.For example, in order to improve the effectiveness of present composition treatment or strengthen another protective effect for the treatment of (the second treatment) vaccine or antimicrobial therapy, may expect these compositionss and method and for example, in other medicaments and the method (antibacterium, antiviral and/or antifungal therapy) for the treatment of, reduction risk of infection or prevent disease and pathogenicity situation combined.

Can apply multiple combination; For example StIR compositions is " A ", and the second treatment is " B ":

A／B／A?B／A／B?B／B／A?A／A／B?A／B／B?B／A／A?A／B／B／B?B／A／B／B

B／B／B／A?B／B／A／B?A／A／B／B?A／B／A／B?A／B／B／A?B／B／A／A

B／A／B/A?B／A／A／B?A/A／A／B?B/A／A／A?A/B/A/AA/A/B/A

Use compositions of the present invention by following the general approach of using by respiratory system, also follow the general approach of using specific the second treatment to experimenter, the toxicity (if there is) of this treatment is taken into account.Estimate repetitive therapy circulation if desired.Also consider that multiple standards is treated and vaccination can be applied with described therapeutic combination.

A. antiviral agent

Of the present invention, aspect some, antiviral agent can use with StIR combination of compositions.Antiviral drugs is the class medicament that specificity is used for the treatment of viral infection, and it should distinguish with antiviral, the antiviral sv virion that initiatively goes out.Available most of antiviral agent are designed to help to process HIV, herpesvirus, hepatitis B and hepatitis C virus at present, and influenza A virus and Influenza B virus.Can be used for antiviral agent of the present invention and include but not limited to immunoglobulin, amantadine, interferon, nucleotide analog and protease inhibitor.

A kind of antiviral strategy is the ability that viral interference is infiltrated target cell.This stage that can use simulated virus to suppress virus replication in conjunction with albumen (VAP) the reagent of being combined with cell receptor.Or by the reagent that uses analog cell receptor and be combined with VAP.This comprises anti-VAP antibody, receptor anti-idiotypic antibody, external receptor and synthetic receptor mimics.Having introduced two kinds of these type of amantadines that " enter blocker " and rimantadine comes influenza.

The second method of antiviral therapy is the process of synthetic virus component after targeting Virus entry cell.A kind of mode of doing is like this exploitation nucleotide or nucleoside analog, and it looks like the construction unit of RNA or DNA, once even if but this analog mixes the enzyme deactivation of synthetic RNA or DNA.Nucleotide analog includes but not limited to ribivirin, vidarabine, acyclovir, gangcyclovir, azidothymidine AZT, Didanosine, zalcitabine, stavudine and lamivudine.

Another antivirus technology is one group of medicine based on ribozyme, and described ribozyme is the enzyme that cuts viral RNA or DNA in selected site.In its natural process, ribozyme is as a part for virus production sequence, but these synthetic ribozymes are designed to cut RNA and DNA on the site that makes its loss of function.

Some viruses comprise the enzyme that is called protease, and described protease cutting virus protein chain, so they can be assembled into final structure.HIV comprises protease, and has carried out large quantity research and found " protease inhibitor ", attacks HIV with this one-phase at its life cycle.Have protease inhibitor to use since nineteen nineties, and proved effectively, but they have uncommon side effect, for example, cause that fat is in uncommon local accumulation.The protease inhibitor improving is just under development at present.

Virus life cycle final stage be from host cell, to discharge complete virus, and antiviral drugs developer also targeting this stage.That has introduced treatment influenza is called zanamivir (RELENZA ^tM) and oseltamivir (TAMIFLU ^tM) two kinds of medicines molecule of being called neuraminidase by blocking-up prevent the release of virion, described neuraminidase is present on the surface of influenza virus, and seems also invariable in a large amount of influenza strains.

Antiviral agent includes but not limited to Abacavir; Acemannan; Aciclovir; Acycloguanosine sodium; Adefovirdipivoxil; Alovudine; Alvircept sudotox; Amantadine Hydrochloride; Amprenavir; Aranotin; Alismone; Ah 's dimension is fixed; Avridine; Cidofovir; Cipamfylline; Cytarabine hydrochloride; Delavirdine mesylate; Descycl; Didanosine; Two

sha Li; Edoxudine; Efavirenz; Enviradene; Enviroxime; Famciclovir; Famotine hydrochloride; Fiacitabine; Fialuridine; Fosarilate; Phosphonoformic acid; Fosfonet sodium; Cymevan; Ganciclovir Sodium; Idoxuridine; Indinavir; U-2032; Lamivudine; Lobucavir; Memotine hydrochloride; Methisazone; Nelfinavir; Nevirapine; Penciclovir; Pirodavir; Ribavirin; Rimantadine hydrochloride; Ritonavir; Ro-31-8959; Somantadine hydrochloride; Sorivudine; Statolon; Stavudine; Hydrochloric acid ladder network dragon; Trifluridine; Valaciclovir hydrochloric acid; Vidarabine; Vidarabine phosphate; Vidarabine phosphate sodium; Viroxime; Zalcitabine; Azidothymidine AZT; Zinviroxime; Interferon, cyclovir, interferon-alpha and/or beta Globulin.

In certain embodiments, antiviral agent is the ribavirin of ribavirin and high dose.Ribavirin is to many DNA and the activated antiviral drugs of RNA viruses.It is the member of nucleoside antimetabolite medicine, the copying of its viral interference hereditary material.Although be not all effective to all virus, ribavirin has broad spectrum of activity, comprises the important activity for the pathogen of influenza virus, arbovirus and many viral hemorrhagic fevers.

Conventionally, the per os form of ribavirin (Ribavirin) and glycol interferon drug regimen are used for the treatment of hepatitis C.Aerosol form has been used for the treatment of respiratory syncytial virus relevant disease in child in the past.But its effect has been queried in multinomial research, and most of mechanism has not re-used it.

B. antibacterial

The example of antibacterial includes but not limited to beta-Lactam antibiotic, penicillin (as, natural penicillin, Aminopenicillin, penicillinase resistance penicillin, penicillin carboxy, urea groups penicillin), cephalosporin (the first generation, the second filial generation and third generation cephalosporin), with other beta-lactams (as, imipenum, monobactam), beta-lactam inhibitor, vancomycin, aminoglycosides and spectinomycin, tetracycline, chloromycetin, Abboticine, lincomycin, clindamycin, rifampicin, metronidazole, polymyxins, sulfa drugs and trimethoprim, and quinoline.Anti-bacterial drug also includes but not limited to: acedapsone, acetosulphone, alamecin, alexidine, Amdinocillin, amdinocillin pivoxil, amicycline, Win-49375, Win-49375, amikacin, amikacin sulfate, aminosallcylic acid, sodium aminosalicylate, amoxicillin, amfomycin, ampicillin, sodium ampicillin, Apalcillin Sodium, aburamycin, aspartocin, astromicin sulfate, avilamycin, avoparcin, Azithromycin, azlocillin, Azlocillin Sodium, Bacampicillin Hydrochloride, bacitracin, bacitracin methylene disalicylate, bacitracin zinc, bambermycin, benzoylpas calcium, erythromycin B, betamicin sulfate, biapenem, biniramycin, biphenamine hydrochloride, bispyrithione magsulfex, Butikacin, butirosin sulfate, capreomycin sulfate Capastat sulfate, carbadox, Carbenicillin Disodium, carindacillin sodium, carbenicillin phenyl sodium, carbenicillin potassium, Carumonam Sodium, cefaclor, cefadroxil, cefadole, Cefadole, cefamandole nafate, Cefaparole, cefatrizine, cefazaflur sodium, cefazolin sodium, cefazolin sodium, cefbuperazone, cefdinir, Cefepime, hydrochloric acid Cefepime, cefetecol, cefixime, Cefinenoxime Hydrochloride, Cefinetazole, Cefinetazole sodium, cefonicid sodium, cefonicid sodium, cefoperazone sodium, ceforanide, cefotaxime sodium, Cefotetan Disodium, Cefotetan Disodium, cefotiam hydrochloride, cefoxitin, cefoxitin sodium, cefpimizole, cefpimizole sodium, cefpiramide, cefpiramide sodium, Cefpirome Sulfate, cefpodoxime, cefprozil, cefroxadine, cefsulodine sodium, ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime, cefuroxime, cefuroxime pivoxetil, Cefuroxime Sodium, celospor, cephalexine, cephalexine hydrochloric acid, Cephaloglycini, cephaloridine, cephalothin sodium, cefapirin sodium, cephradine, cetotetrine hydrochloride, cetophenicol, chloromycetin, Cliloramphenicol Palmitate, Chloramphenicol Pantotheniate Complex, chloramghenicol sodium succinate, chlorhexidine phosphanilate, chlorxylone, chlortetracycline bisulfate, chlortetracycline hydrochloride, cinoxacin, ciprofloxacin, ciprofloxacin, Cirolemycin, Clarith, AM-1091, Clildamycin, Clindamycin Hydrochloride, clindamycin palmitate hydrochloride, cleocin phosphate, clofazimine, benzathine cloxacillin, cloxacillin sodium, cloxiquine, polymyxin e methanesulfonic sodium, polymyxin E sulfate, coumamycin, coumamycin sodium, cyclacillin, cycloserine, dalfopristin, dapsone, daptomycin, Demeclocycine, Demeclocycine Hydrochloride, demecycline, denofungin, diaveridine, dicloxacillin, dicloxacillin sodium, dihydrostreptomycin sulfate, dipyrithione, dirithromycin, doxycycline, doxycycline calcium, doxycycline fosfatex, doxycycline hydrochloride, droxacin sodium, ENOXACIN, dexacillin, epitetracycline hydrochloride, Abboticine, Erythromycin Acistrate, erythromycin propionate lauryl sulfate, erythromycin ethylsuccinate, erythromycin gluceptate, Erythromycin Lactobionate, erythromycin propionate, bristamycin, ebutol, ethionamide, Fleroxacin, flucloxacillin, fludalanine, flumequine, fosfomycin, fosfomycin trometamol, fumoxicillin, furazolium chloride, furazolium tartrate, sodium fusidate, fusidic acid, Gentamicin Sulfate, gloximonam, Gramicidin, haloprogin, Hetabiotic, Hetacin-K (Fort Dodge), hexedine, ibafloxacin, ibafloxacin, isoconazole, isepamicin, isoniazid, EN-141, kanamycin sulfate, kitasamycin, levofuraltadone, phenoxypropyl penicillin potassium, Lexithromycin, lincomycin, lincomycin hydrochloride, Bareon, hydrochloric acid Bareon, Lomefloxacin Mesylate, Lorabid, mafenide, meclocycline, meclocycline sulfosalicylate, megalomicin potassium phosphate, mequidox, doubly can, methacycline, methacycline hydrochloride, hexamethylenamine, methenamine hippu, metronidazole, Mezlo, mezlocillin sodium, minocycline, minocycline hydrochloride, mirincamycin hydrochloride, monensin, monensin sodium, sodium nafcillin, Nalidixate Sodium, nalidixan, pimaricin, tobramycin, neomycin palmitate, polygynax, neodecyllin, Netilmicin Sulfate, neutramycin, nifuradene, nifuraldezone, magmilor, nifuratrone, nifurdazil, nifurimide, nifurquinazol, nifurthiazole, nitrocycline, nitrofurantoin, Nitromide, norfloxacin, novobiocin monosodium, ofloxacin, ormetoprim, oxacillin sodium, oximonam, oximonam sodium, oxolinic acid, geomycin, Calcium Oxytetracycline., tetramycin hydrochloride, paldimycin, parachlorophenol, paulomycin, pefloxacin, penamecillin, benzathine penicillin G, scotcil, procaine benzylpenicillin, penicillin G sodium, penicillin V, penicillin V benzathine, abbocillin V, potassium v calcium, pentizidone sodium, tebamin, avocin, pirbenicillin sodium, piridicillin sodium, pirlimycin hydrochloride, pivampicillin hydrochloride, pivampicillin pamoate, Pivampicillin Probenate, aerosporin, porfiromycin, propikacin, pyrazinamide, vancide ZP, quindecamine acetate, quinupristin, racefenicol, Ramoplanin, ranimycin, relomycin, repromicin, Mycobutin, rifametane, rifamexil, rifamide, rifampicin, rifapentine, rifapentine rifaximin, pyrrolidinomethyl tetracycline, rolitetracycline nitrate, rosamicin, rosamicin butyrate, rosamicin propionate, rosamicin sodium phosphate, rosamicin stearate, rosoxacin, ristal, Roxithromycin, bonomycin, sanfetrinem sodium, Oxetacillin, Sarpicillin, scopafungin rickamicin, Sisomicin, sisomycin sulfate, Sparfloxacin, spectinomycin hydrochloride, acetylspiramycin, stallimycin hydrochloride, steffimycin, streptomycin sulfate, streptoniazide, sulfabenz, sulfbenzamide, sulfacetamide, sulphacetamide, renoquid, sulfadiazine, sulfadiazine sodium, fanasil, sulfalene, sulfamethyldiazine, sulfanilamide (5) is to Sulfamonomethoxine, sulfamethazine, ayerlucil, sulfamethoxine

azoles, sulfamonomethoxine, sulfanilamide diformazan

azoles, sulfanilate zinc, sulfanitran, sulfasalazine, amidozoi, ST, sulfamethylphenazole, sulfanilamide are different

azoles, Sodium Sulfacetamide are different

azoles, sulfanilamide are different

azoles diethanolamine salt, sulfomyxin, sulopenem, sulbactam, suncillin sodium, Talampicillin Hydrochloride, teicoplanin, hydrochloric acid Temafloxacin, temocillin, tetracycline, quadracycline, Telotrex (Bristol-Myers Squibb), tetroxoprim, thiamphenicol, potassium thiphencillin, Ticarcillin Cresyl Sodium, ticarcillin disodium, ticarcillin sodium, 1443 ticlatone, tiodonium chloride, tobramycin, tobramycin sulfate, A-6096, trimethoprim, trimethoprim sulfate, neotrizine, triacetyloleandomycin, trospectomycin sulfate, Tyrothricin, vancomycin, Lyphocin (Fujisawa), virginiamycin and/or laramycin.

B. antifungal

Antifungal includes but not limited to azole, imidazoles, polyene, posaconazole, fluconazol, Itraconazole, amphotericin B, 5-flurocytosine, miconazole, ketoconazole, ethambutol (ebutol), dapsone (4,4 '-diaminourea hexichol phenylsulfone), Paser granule (aminosallcylic acid granule), rifapentine, pyrazinamide, isoniazid, rifampicin IV, rifampicin, pyrazinamide, streptomycin sulfate, ethionamide-SC (ethionamide) and/or voriconazole (Vfend ^tM).

C. other medicaments

Of the present invention, aspect some, antiinflammatory can use with StIR combination of compositions.

Include but not limited to Fluticasone, beclometasone, its any pharmaceutically acceptable derivant for steroidal anti-inflammatory agents herein, and any combination.As used herein, pharmaceutically acceptable derivant comprises its any salt, ester, enol ether, enol ester, acid, alkali, solvate or hydrate.Can use this type of derivative known method to prepare this analog derivative by those skilled in the art.

Fluticasone. fluticasone propionate is synthetic corticosteroid the formula C that sees service ₂₅h ₃₁f ₃o ₅s.Its chemistry S-(methyl fluoride) 6 α by name, Alpha-Methyl-3-oxo androstane-Isosorbide-5-Nitrae-diene-17,9-bis-fluoro-11 β-17-dihydroxy-16 β-thiocarboxylic, 17-propionic ester.Fluticasone propionate is to have the white of 500.6 molecular weight to pale powder, substantially insoluble in water, is soluble in dimethyl sulfoxine and dimethyl formamide, slightly soluble in methanol and 95% ethanol.

In one embodiment, preparation of the present invention can comprise steroidal anti-inflammatory agents (for example, fluticasone propionate).

Beclometasone. in some aspects, steroidal anti-inflammatory agents can be beclomethasone dipropionate or its monohydrate.The chemistry chloro-11b of 9-by name of beclomethasone dipropionate, pregnant steroid-Isosorbide-5-Nitrae-diene-3 of 17,21-trihydroxy-16b-methyl, 20-diketone 17,21-dipropionate.This compound can be that molecular weight is 521.25 white powder; And soluble,very slightly in water (Physicians ' Desk Reference), in chloroform, very easily dissolve, and soluble in acetone and alcohol.

Provide steroidal anti-inflammatory agents of the present invention to strengthen the compositions and methods of the invention by for example alleviating any undesired inflammation.Example for other steroidal anti-inflammatory agents herein includes but not limited to betamethasone, omcilon, dexamethasone, prednisone, mometasone, flunisolide and budesonide.

According to another aspect of the invention, non-steroidal anti-inflammatory agents can comprise aspirin, sodium salicylate, acetaminophen, Phenacetin, ibuprofen, ketone ibuprofen, indomethacin, BTS-18322, diclofenac, naproxen, piroxicam, Tebufelone, etodolac, Nabumetone, tenidap, alcofenac, phenazone, amimopyrine, dipyrone, animopyrone, Phenylbutazone, clofexan, crovaril, prexazone, azapropazone, benzydamine, BCP, cinchophen, clonixin, ditrazol, epirizole, fenoprofen, floctafeninl, flufenamic acid, glafenine, Indoprofen, meclofenamic acid, mefenamic acid, niflumic acid, salidifamides, sulindac, suprofen, Tolmetin, Nabumetone, FK-1160, proquazone, bufexamac, flumizole, tienoridine, timegadine, dapsone, Diflonid, benorylate, fosfosal, fenclofenac, etodolac, Fentiazac, tilomisole, Carprofen, fenbufen,

promazine, Artiflam, pirprofen, Feprazone, piroxicam, sudoxicam, isoxicam, celecoxib,

and/or tenoxicam.

VII. medicine box

Any compositions described herein can be included in medicine box.In a limiting examples, for generation of and/or the reagent of sending StIR compositions be included in medicine box.In some aspects, described medicine box is portable, and can as asthma inhaler, carry.Described medicine box also can comprise pathogen detection device.Described medicine box also can be containing the gas or the mechanical propellant that are useful on the present composition.

Component in described medicine box can water, powder or lyophilized form packing.The container of medicine box generally comprises at least one inhaler, tank, pipe, test tube, flask, bottle, syringe or other containers, wherein can place component the component of preferably placing suitable decile.While containing more than a kind of component in medicine box (the second medicament etc.), described medicine box generally also contains second, the 3rd or other extra containers, wherein can place separately extra component.But, can in pipe, tank or inhaler, comprise various ingredients combination.Container of the present invention can comprise tank or inhaler, and it can be through on belt or easily be carried in pocket, knapsack or other storage capsules.Medicine box of the present invention also comprises conventionally for the container of described compositions or its variant, and close packing is for any reagent container of commercialization sale.This type of container can comprise the plastic containers of injection moulding or blowing, is wherein fixed wtih the pipe of wanting.

When provide the component of medicine box in a kind of and/or plurality of liquid solution time, for example liquid solution is aqueous solution, especially preferably aseptic aqueous solution (but not necessary).But the component of medicine box also can provide by dry powder.In the time that reagent and/or component provide with dry powder, can again dissolve powder or use with powder form by adding suitable solvent.Solvent also can be considered to provide in another container.

Medicine box also comprises the description that uses kit components and use any other reagent not to be covered in medicine box.Description can comprise changing form of can implementing.

Consider that this type of reagent is the embodiment of medicine box of the present invention.But, the specific project that this type of medicine box is not limited to above identify, and can be included in any reagent directly or indirectly using in the using of the detection of pathogenic microbes or StIR compositions of the present invention.

VIII. embodiment

Provide following examples for illustrating the multiple embodiment objects of the present invention, and it is not intended to limit in any form the present invention.Those skilled in the art can easily understand, and the present invention is very suitable for realizing object and obtains mentioned target and advantage, and intrinsic those objects, target and the advantage of this paper.The embodiment of the present invention and method described herein represent some embodiment, are not intended to limit the scope of the invention.Those skilled in the art can expect other purposes that comprise in the spirit of the present invention of change wherein and claim scope definition.

Embodiment 1

Pseudomonas aeruginosa is attacked.Obtain bacterial strain PA103 and in 20% glycerol, save as refrigerated storage liquid (1 × 10 LB culture medium (Bio101Systems) from ATCC ⁸cFU/ml).By 1ml stock solution 5%CO at 37 ℃ ₂in middle 100ml LB culture medium, hatch 16 hours, then in 1L fresh medium, dilute, and cultivate 6-7 hour at 37 ℃, to OD ₆₀₀reach 0.3, produce～3 × 10 ¹⁰cFU.Centrifugal suspension, washing, resuspension atomization are attacked, by by serial dilutions bed board to the concentration of measuring antibacterial on tryptic soy agar plate (Becton Dickinson).For carrying out atomization, 10ml suspension is placed in by 10L/ minute air 5%CO ₂in the AeroMist CA-209 aerosol apparatus (CIS-US) driving, to promote degree of depth ventilation.After 30 minutes, then add 5ml, make during whole 60 minutes 10ml suspension altogether be atomized.

The processing of TLR part.Before infecting and attacking, with the aerosol (alone or in combination) of TLR part or with PBS (negative control) processing mice.Before infecting attack, send all processing 18 hours, use and be supplemented with 5%CO ₂10L/ minute drive AeroMist CA-209 aerosol apparatus with promote ventilate.Process for each, TLR part suspension or the PBS of 10ml are placed in to aerosol apparatus, and used in 20 minutes.For the experiment that uses TLR ligand combination, two kinds of parts are suspended in same 10ml suspension, and send simultaneously.For each part, the minimum suspension concentration infiltrating by the neutrophil cell of induction lung is determined initial aerosol agent dose, and this concentration is counted to measure by processing leukocyte and neutrophil cell total in latter 24 hours bronchoalveolar lavage fluid.

TLR4 part.Different from the natural lipid A of the mixture that contains 5,6 and 7 acyl groups, LA-synthetic (MPLA, Invivogen) is the pure synthetic that contains 6 fatty acyl groups.Send the suspension of MPLA with 100 μ g/ml.Send another synthetic lipid A phosphorylation six acyl group disaccharide (PHAD, Avanti Polar Lipids) one by one with 6 fatty acyl groups with 100 μ g/ml.

TLR2/6 part.Pam2CSK4 and FSL-1 (both are all from Invivogen) are the synthesis of diacyl lipopeptids that the known heterodimer by TLR2 and TLR6 carries out signal transduction.As shown, send Pam2CSK4 with 6 or 20 μ g/ml, and send FSL-1 with 20 μ g/ml.

TLR9 part.ODN2395 (Invivogen) is C type CpG ODN people and Mus TLR9 to high-affinity.With 20 μ g/ml atomization ODN2395.

TLR7 part.Imiquimod (R837, Invivogen) is the imidazole quinoline amine guanosine analogue that stimulates TLR7 (also may stimulate TLR8).As shown, send imiquimod with the aerosol of 1 or 300 μ g/ml.

TLR5 part.The conservative 22 aminoacid sections (known TLR5 part) of flagellin camber are identified.This aminoacid section is at Cell Essentials, Inc., and Boston, MA synthesizes.Based on the purity > 95% of HPLC and Malidi-TOF mass spectrum checking peptide.Send the synthetic fragment of Flg22 with 100 μ g/ml.

Embodiment 2

Materials and methods

Animal and reagent.All general reagent all obtains from Sigma (St Louis, MO), except as otherwise noted.Show loving care for and use the policy of committee to process all mices according to M.D.Anderson Cancer center of University of Texas animal.The wild females Swiss-Webster mice in 5 to 8 week age (Charles River, Wilmington, MA) is tested for great majority protection and cell counting.As shown, the female MyD88 in 5 to 8 week age Shizuo Akira (1998) being provided ^-/-mice, Trif ^-/-mice (The Jackson Laboratory, Bar Harbor, ME) and TLR2 ^-/-mice (Jackson) is for comparing with wild-type mice C57BL/6J (Jackson).

Atomization processing.At chocolate agar (Remel, Lenexa, KS) the refrigerated storage liquid that upper cultivation can not typing hemophilus influenza (NTHi), at the brain heart infusion culture fluid (Acumedia that is supplemented with 3.5 μ g/ml NAD, Baltimore, MD) in amplification, and as described in (Clement etc., 2008; Evans etc., 2010; Moghaddam etc., 2008) use EmulsiFlex C5 (Avestin, Mannheim, Germany) to carry out fragmentation.Measure (Pierce, Rockford, IL) by bicinchoninic, in saline, protein concentration is adjusted to 2.5mg/ml, and lysate is chilled in to-80 ℃ with 10ml aliquot.For processing, the aliquot of thawing is placed in and is supplemented with 5%CO ₂in 10l/ minute air operated AeroMist CA-209 aerosol apparatus (CIS-US) of (promote deeply breathe) 20 minutes.Aerosol apparatus is connected in 10 liters of polyethylene contact chambers by polyethylene tube (30cm x22mm), there is low resistance microbe filter (BB50T, Pall, East Hills at its end, NY) identical outflow pipeline, is discharged in Biosecurity hood.

Buy Pam3CSK4, Pam2CSK4, Poly (I:C), MPLA, imiquimod and ODN2395 from InvivoGen (San Diego, California).By the Flg22 of the synthetic 22-mer of Cell Essentials (Boston, MA), its conserved domain (QRLSTGSRINSAKDDAAGLQIA) that is flagellin.In order to process mice, in without endotoxic water, again dissolve synthetic TLR agonist, and with shown in concentration be suspended from the aseptic PBS of 8ml, and use and animal carried out to atomization 20 minutes for the constructed of NTHi lysate processing.

In vivo infection is attacked.(Clement etc., 2008 as described previously; Clement etc., 2009; Evans etc., 2010), with determining to LD ₈₀-LD ₁₀₀bacterial inoculum suck attack mice.Obtain Pseudomonas aeruginosa bacterial strain PA103 and save as refrigerated storage liquid (1 × 10 20% glycerol of LB culture medium (Bi0101Systems) from ATCC ⁸cFU/ml).5%CO at 37 ℃ ₂in, 1ml stock solution is hatched 16 hours in 100ml LB culture medium, then in 1l fresh medium, dilute, and cultivate 6-7 hour at 37 ℃, to OD ₆₀₀reach 0.3, produce 1-4 × 10 ¹⁰cFU/ml.Streptococcus pneumoniae serotype 4 is saved as to refrigerated storage liquid (1 × 10 in 20% glycerol of Todd-Hewett culture fluid (Becton Dickinson) ⁹cFU).5%CO at 37 ℃ ₂in, the stock solution that 1ml is thawed is hatched 16 hours in 150ml Todd-Hewett culture fluid, then in 1.5l fresh medium, dilutes, and cultivate 6-7 hour in logarithmic (log) phase, to OD ₆₀₀reach 0.3, produce 2-6 × 10 ¹⁰cFU/ml.By centrifugal bacterial suspension, wash, be resuspended in 10ml PBS, and use with for the treatment of the identical system of system in 60 minutes, carry out atomization.By at tryptic soy agar plate (Becton Dickinson), above serial dilution is carried out to bed board measures bacterial concentration.

It is quantitative that pneumonopathy substance is loaded.(Clement etc., 2008 as discussed previously; Clement etc., 2009; Evans etc., 2010), anesthetized mice immediately after infecting with bacterial pathogens, collects their lung, and uses the tissue grinder (Kontes, Vineland, NJ) of 2ml in 1ml PBS, to carry out homogenate.On tryptic soy agar (TSA) flat board, the homogenate of serial dilution is carried out to bed board, at 37 ℃, hatch 16 hours, and enumeration of bacterial colonies.

BALF Analysis.(Clement etc., 2008 as discussed previously; Clement etc., 2009; Evans etc., 2010), by instiling and expose the large size source nipple intubate two parts of aliquots of collection (each 1ml PBS) (Becton Dickinson) in the ring of conduit by insertion on the time point of specifying, obtain bronchoalveolar lavage fluid (BAL).Utilize hematimeter (Hauser Scientific, Horsham, PA), and by the BAL fluid differential count of 5 minutes at 2,000rpm cell centrifugation, 300 μ l, Yong Lai-Ji dyes to measure total quantity of leucocyte subsequently.

The external mensuration of killing and wounding.(Clement etc., 2008 as discussed previously; Clement etc., 2009; Evans etc., 2010), in 6 orifice plates, be supplemented with in the RPMI-1640 of 10% hot deactivation FCS and 1% penicillin/streptomycin (Invitrogen) and cultivate MLE-15 cell and A549 cell.When growing to～80% while converging, use PBS washed cell, the fresh antibiotic-free culture medium that contains 10% hot deactivation FCS is provided, and processes with ODN2395 (20 μ g/ml), the Pam2CSK4 (10 μ g/ml) of 20 μ l volumes in 20 μ lPBS or the RPMI-1640 that contains 10% hot deactivation FCS or ODN2395 (20 μ g/ml) and Pam2CSK4 (10 μ g/ml).After 4 hours, then 1000 spore of Bacillus anthracis Sterne bacterial strain or 2000CFU Pseudomonas aeruginosa bacterial strain PA103 are added to institute porose in.Infect latter 4 hours, sucking-off 20 μ l supernatant from each hole, serial dilution, tiling, to TSA agar plate, is hatched 16 hours, and is counted CFU at 37 ℃.

Immunofluorescence microscopy.At Lab-Tek II chamber microscope slide (Nunc, Rochester, NY) on, be supplemented with in the RPMI-1640 of 10% hot deactivation FCS and 1% penicillin/streptomycin (Invitrogen) and cultivate A549 cell 48 hours, then use ODN2395 (the 20 μ g/m of the Texas Red-labelling of 20ml volume in the RPMI-1640 that contains 10% hot deactivation FCS, Invivogen), Pam2CSK4 (the 10 μ g/ml of Fluorescein isothiocyanate (FITC) labelling, or the ODN2395 of Texas Red-labelling (20 μ g/ml Invivogen), and Pam2CSK4 (the 10 μ g/ml of Fluorescein isothiocyanate (FITC) labelling Invivogen), Invivogen) process.After 2 hours, draw culture medium, separate cell, and with ice-cold PBS washed cell 3 times.Then use 4% paraformaldehyde fixed cell, use glycine quencher, with PBS washing 3 times, with 4 ', 6-bis-narrows base-2-phenindione (DAPI; 0.1 μ g/ml) redye nucleus, and utilize fluorescence microscopy (Olympus BX-60microscope, Melville, NY) to use suitable optics (Texas Red: excite=540nm; Transmitting=620nm; FITC: excite=495nm transmitting=520nm; DAPI: excite=360nm; Transmitting=460nm) check.The Spot RT Camera (Diagnostic Instruments, Sterling Heights, MI) computerizeing control collects image and assembling in Photoshop CS3 (Adobe, San Jose, CA) continuously.Overlapping redness and green fluorescence are rendered as yellow.

Statistical analysis.Use SAS/STAT software (version 8.2, SAS Institute) to carry out statistical analysis.Student ' s t-check is used for to lung antibacterial or the virus titer between comparable group.Use the relatively percentage ratio of the rear survival mice of pathogen attack of Fisher ' s Precision Test, and log-rank check distributes for the survival of relatively estimating by Kaplan-Meier method.Single tail ANOVA and Dunnett ' s check for relatively processing the BAL fluid differential count between untreated animal afterwards.

Result

That the induction of atomization antibacterial lysate needs the resistance of pneumonia is MyD88 rather than TRIF.Stimulate lung epithelial to induce high-level resistance (Clement etc., 2008 to extensive microbial pathogens 9 by the atomization lysate of NTHi; Clement etc., 2009; Evans etc., 2010; Tuvim etc., 2009).Be whether that the protection of lysate induction is required in order to test TLR signal transduction, suck to attack with Pseudomonas aeruginosa to lack the mice that carries out TLR signal transduction by TIR adaptin.Carry out pretreatment by atomization antibacterial lysate, wild type and TRIF deficiency (Trif ^-/-) mice protected completely for the infection of lethal Pseudomonas aeruginosa, and at MyD88 deficient mice (Myd88 ^-/-; FIG.12A and 12B, left figure) in can not reactance power.Protection kills and wounds closely related (FIG.12A and 12B, right figure) with the quick pathogen in induction lung.IL-1 receptor also carries out signal transduction (Adachi etc., 1998 by MyD88; Medzhitov etc., 1998), but it responds to host cell factor signaling, and directly do not respond to microbial product.Pathogen is killed and wounded the IL-1 receptor defects type mice (Il1r stimulating being atomized antibacterial lysate ^-/-; FIG.13) in, retain completely.This discovery shows; it is required not to be that all receptors of carrying out signal transduction by MyD88 are the protection of lysate induction; and prompting, the direct microorganism signal transduction being undertaken by TLR is more even more important for inductivity epithelium resistance than the indirect signal transduction by the host cell factor.

The low-level resistance of individual TLR agonist induction to pneumonia.In view of the demand to MyD88 signal transduction, whether the inventor has tested any single synthetic TLR agonist the similar resistance of resistance providing to atomization antibacterial lysate can be provided.Because TLR1 and TLR6 are expressed as the heterodimer with TLR2, and TLR7 and TLR8 all identify imiquimod, so mice TLR1 to 9 all available following 7 kinds of synthetic ligands stimulates: Pam3CSK4 (TLR2/1 agonist), Pam2CSK4 (TLR2/6 agonist), Poly (I:C) (TLR3 agonist), synthetic lipid A (MPLA, TLR4 agonist), Flg22 (synthetic 22-mer flagellin, TLR5 agonist), imiquimod (TLR7 and TLR8) or ODN2395 (TLR9 agonist).

The suitable atomization dosage of these agonist is unknown, so formulated strategy to identify suitable dosage, for send to avoid the mistake of II (β) type to lung.Each synthetic TLR agonist used all has the report concentration ([DCmax]) (Yamamoto etc., 2003 that stimulate the secretion of the dendritic cell maximum cell factor; Aliprantis etc., 1999; Buwitt-Beckmann etc., 2005; Hayashi etc., 2001; Krug etc., 2001; Lee etc., 2003; Martin etc., 2003).Calculating (Clement etc., 2009 based on effective air flue of atomization compound is sent; Evans etc., 2004), the inventor has measured and on airway epithelia surface, has realized the aerosol apparatus fluid concentrations that [DCmax] needs.Although not relying on leukocyte, the resistance of atomization lysate induction flows into cycle and the intensity of the lung neutrophil increase disease of protection phenomenon and induction be closely related (Clement etc., 2008).Therefore,, in order to identify the TLR agonist dosage that is enough to be used in test, the inventor is from the report [DCmax] of each part, and logarithm increase spraying concentration, until realize leukocyte infiltration.

As shown in Figure 14, in the mice of processing at PBS, in BAL liquid, the quantity of neutrophil cell is 0.1 × 10 ³± 0.2 cell/ml.Only Pam2CSK4 shows the remarkable increase of neutrophil cell at DCmax place, although all show the remarkable increase of neutrophil cell level at the concentration place higher than one or two logarithm of DCmax except poly (I:C) and Flg22.On the other hand, processing after 24 hours, poly (I:C) and Flg22 all induce macrophage to flow in a large number on BAL.Flg22 and imiquimod have such ligand concentration separately, infiltrate and reduce higher than described concentration place neutrophil cell.The neutrophil cell level that Pam2CSK4 induction is nearly 5 times of Pam3CSK4, and be 5 times of any other ligand 1s.

The selected concentration of each part is that 10 times of induction neutrophil cell/ml increases or induction macrophage are doubled the lowest dose level of (can simultaneously realize the two without any).Some parts are induced strong cellular infiltration, protect (Figure 15) but do not have a kind of synthetic agonist to provide to try hard to keep for lethal Pseudomonas aeruginosa pneumonia.Pam2CSK4, Flg22 and imiquimod have protection tendency, but these all do not reach the significance,statistical of single experiment or many experiments meansigma methods.The mice that MPLA the processes non-remarkable trend that show needle improves mortality rate after pathogen is attacked.

The high-level resistance to pneumonia has been induced in the combination of TLR2/6 and TLR9 agonist.Although single synthetic TLR agonist only provides moderate protection, likely stimulate when multiple PRR as induction high-level resistance required (Clement etc., 2008; Evans etc., 2010).Whether can induction of resistance for the combination of determining TLR agonist, the inventor has tested the pairing of seven kinds of synthetic ligands and has arranged.

It should be noted that, utilize Pam2CSK4 and ODN2395 (ODN+Pam2) to process to cause 100% mice in other cases for the Gram-negative Pseudomonas aeruginosa of lethal is attacked lower survival (Figure 16 A simultaneously, left), with 80% mice survive under the lethal hit of Gram-positive streptococcus pneumoniae (Figure 16 B, a left side).The concentration of two kinds of parts is doubled to cause 90% survival (Figure 16 B) under streptococcus pneumoniae is attacked in aerosol is processed.Protection mice avoids lethal infection to be attacked and the Synergistic killing relevant (Figure 16 A and 16B, the right side) of the interior pathogen of lung, and it is relevant that ligand concentration is doubled to the tendency of killing and wounding to higher pathogen.At 4 hours and 24 hours, at leukocyte to the cooperative interaction (Figure 16 C) of having observed Pam2CSK4 and ODN2395 in the raising of lung.These results show, the part induction antimicrobial effect of TLR2/6 and TLR9 is replied the collaborative activation of (comprise pathogen is killed and wounded with those of leukocyte recruitment reply), and it causes the collaborative level for the protection of pneumonia.Similar with the kinetics of the resistance of NTHi lysate induction, within latter 4 hours, there is protection in processing.

Not every TLR agonist combination all produces for the strong protection of infecting.The inventor has tested the following combination of TLR agonist: Pam2+Poly (I:C), Pam2+Flg22, Pam2+ imiquimod, ODN+Poly (I:C), ODN+Flg22 and ODN+Pam3.The inventor finds that these combinations are compared with Pam2-ODN combination (Figure 16), effect lower (Figure 17 A-F) in the time protecting for Pseudomonas aeruginosa.These results disclose, and the immunostimulation identical with Pam2-ODN all given in not every TLR agonist combination.

TLR2 is enough to promote protectiveness Pam2SCK4 and ODN2395 concertedness, but is not that reactance power is necessary.The presumptive evidence that the detection of the cooperative effect of TLR part Pam2SCK4 and ODN2395 (it has the receptor-specific of abundant definition) provides TLR2/6 and TLR9 to participate in.The inventor uses knock-out mice and other parts to find extra evidence.

The inventor has compared the survival with Pam2-ODN or the pretreated wild type of PBS and TLR2 deficient mice before Pseudomonas aeruginosa is attacked.Wild-type mice is protected completely by Pam2-ODN, but the wild type group or the arbitrary Tlr2 that process at sham ^-/-not survival (Figure 18 A, left figure) in group, prove needs TLR2 in the protection of Pam2-ODN induction.Tlr2 ^-/-in mice, lack protection and kill and wound closely related (Figure 18 A, right figure) with the interior pathogen of the lung that lacks Pam2-ODN induction.

Because Pam2CSK4 and Pam3CSK4 distinguish TLR2/6 and TLR2/1, and while combining with ODN2395, Pam2CSK4 produces strong coordinating protection effect and Pam3CSK4 is quite different, so may need TLR2/6 heterodimer to induce lung epithelial resistance.The inventor also attacks Tlr2 after NTHi lysate is processed ^-/-and wild-type mice, and find forfeiture (Figure 18 B, left figure) of protection, do not lose the antibacterial of lysate induction yet and kill and wound (Figure 18 B, right figure).In a word, these results suggest, TLR2/6 is enough to the cooperative interaction with TLR9, but the lung epithelial resistance of not all induction is necessary.

C type CpG ODN and Pam2SCK4 cooperative interaction and induce the resistance to bacterial pneumonia, and A type and Type B are quite different.Whether the inventor attempts further to assess TLR9 is required with the cooperative interaction of Pam2-ODN.Because there is no Tlr9 ^-/-mice can be used, so the inventor is with the known participation of not testing TLR9 in conjunction with the out of order ODN of TLR9.Survive with the mice 90% that Pam2-ODN pretreatment causes Pseudomonas aeruginosa to be attacked, but cause not surviving (Figure 19 A) during with Pam2SCK4 and contrast ODN pretreatment, show ODN and TLR9 to be combined into coordinating protection required.

In order further to explore the interactional specificity of Pam2-ODN, the inventor processed wild-type mice with Pam2CSK4 and dissimilar CpG ODN before attacking with Pseudomonas aeruginosa.A type ODN (ODN1585 or ODN2216) or Type B ODN (ODN2006-G5) can not give protection with the combination of Pam2SCK4, and the combination of Pam2SCK4 and C type ODN (ODNM362 or ODN2395) promotes the remarkable resistance (Figure 19 B) to other lethal pneumonia.These results show, TLR2/6 and not only synergism of TLR9 part, and also specific part is more conducive to interact than other parts.

Pam2CSK4 and ODN2395 induce antibacterial to kill and wound by epithelial cell in vitro.In the time stimulating with NTHi lysate, pulmonary epithelial cells is induced to kill antibacterial (Clement etc., 2009; Evans etc., 2010).Because Pam2-ODN has reappeared the immune-stimulating effect of antibacterial lysate in vivo, whether the inventor has tested described combination and also can induce in vitro the pulmonary epithelial cells of separation to carry out pathogen to kill and wound.With Pam2-ODN pretreatment of mice MLE-15 airway epithelial cell 4 hours remarkable reduce the antibacterial CFU (Figure 20 A) in the cell culture medium after inoculation Bacillus anthracis.Similarly, cause infecting the remarkable minimizing (Figure 20 C) of latter 4 hours Pseudomonas aeruginosa CFU with Pam2-ODN handler A549 cell.Prove that it is by stimulating epithelial cell that pathogen is killed and wounded, rather than by the direct antibiotic effect of Pam2-ODN, antibacterial is containing growing to equal amount in epithelial hole, no matter they are process or process (Figure 20 B and 20D) with PBS with Pam2-ODN.

Therefore, in mice and HEP, all induce antimicrobial effect, and caused killing Gram-positive and gram negative bacteria pathogen.These digital simulations in vivo Pam2-ODN process after the antibacterial that observes kill and wound.Continuous raising Pam2-ODN dosage does not significantly improve pathogen to 32 times shown in this article and kills and wounds.

Pam2CSK4 and ODN2395 locate in cell in vivo altogether.Pam2CSK4 and ODN2395 interact and induce synergistic mechanism still not solve.Be positioned on plasma membrane because TLR2/6 is in the news, and TLR9 is in the news and is positioned at (Beutler, 2009 in endosome; Dostert etc., 2008), so people cannot predict the Physical interaction of described part.But, because TLR4 may need internalization to carry out signal transduction (Kagan etc., 2008), so whether the inventor has studied these two parts by epithelial cell internalization.Monolayer culture A549 cell on cell culture microscope slide, is then used in the Pam2CSK4 (10 μ g/ml) of the FITC labelling of the same concentrations using in pathogen killing experiments and the ODN2395 (20 μ g/ml) of Texas Red labelling processes.After 2 hours, washed cell, with DAPI labeled cell core, and with microscope slide described in fluorescence microscope.Pam2CSK4 and ODN2395 are all by epithelial cell internalization.In addition Pam2CSK4 and ODN2395 indoor in cytoplasmic region (inferring in endosome) location altogether.These results suggest Pam2CSK4 and ODN2395 may locate altogether in endosome.

Utilize the combination of Pam2CSK4, TLR2/6 agonist and C type ODN (2395,10101 or M362), the TLR9 agonist high-level resistance to Bacillus anthracis and influenza virus pulmonary infection of having carried out pretreatment induction.Attacking or utilize influenza virus aerosol to attack first 1 day with anthrax spore intranasal, use as shown the TLR part pretreatment of mice of atomization.Monitor the survival of mice.

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Claims (17)

1. treat, suppress or alleviate the method for infected by microbes, it comprises to suffering from infected by microbes or having the individuality that occurs or obtain the risk of infected by microbes to use TLR9 agonist and the TLR2/6 agonist of effective dose.

2. the process of claim 1 wherein that described TLR2/6 agonist is PAM2CSK4.

3. the process of claim 1 wherein that described TLR9 agonist is C type oligodeoxynucleotide (ODN).

4. the method for claim 3, wherein said C type ODN is ODN2395 or ODNM362 or ODN10101.

5. the process of claim 1 wherein that described experimenter has contacted pathogenic microbes.

6. the process of claim 1 wherein that described microorganism is virus, antibacterial or fungus.

7. the method for claim 6, wherein said virus is Adenoviridae, coronaviridae, filamentous virus section, flaviviridae, Hepadnaviridae, herpetoviridae, orthomyxoviridae family, Paramyxoviridae, Pneumovirinae, Picornaviridae, Poxviridae, Retroviridae, Togaviridae, parainfluenza virus, influenza virus, H5N1, Marburg virus, Ebola virus, Severe acute respiratory syndrome coronavirus, yellow fever virus, human respiratory syncytial precursor virus, Hantaan virus or vaccinia virus.

8. the method for claim 6, wherein said antibacterial is Bacillus anthracis (Bacillus anthracis), Yersinia pestis (Yersinia pestis), soil draws hot Frances Salmonella (Francisella tularensis), Pseudomonas aeruginosa (Pseudomonas aerugenosa), staphylococcus aureus (Staphylococcus aureas), pneumonia staphylococcus (Staphylococcus pneumonia), have a liking for maltose Stenotrophomonas (Staphlyococcus maltophilia), bulkholderia cepasea (Burholderia spp.) or catarrhalis (Moraxella spp.).

9. the method for claim 6, wherein said fungus is aspergillosis, candida mycoderma, cryptococcus, histoplasma capsulatum, coccidioides immitis, blastomyces, zygomycete or lung sac worm.

10. the process of claim 1 wherein that described TLR9 agonist and described TLR2/6 agonist use with spray agent.

11. the process of claim 1 wherein that the TLR9 agonist of described effective dose and TLR2/6 agonist are deposited in the lung of described individuality.

12. the process of claim 1 wherein that described TLR9 agonist and described TLR2/6 agonist use to the amount of about 100mg/kg whose body weight with about 0.1mg/kg.

13. pharmaceutically acceptable compositionss, it comprises TLR9 agonist and TLR2/6 agonist, antiinflammatory and one or more pharmaceutical excipients, and wherein said compositions is aseptic and does not substantially contain pathogenic microbes.

The compositions of 14. claim 13, wherein said compositions is mixed with for spraying.

The compositions of 15. claim 13, wherein said TLR2/6 agonist is PAM2CSK4.

The compositions of 16. claim 13, wherein said TLR9 agonist is C type oligodeoxynucleotide (ODN).

The compositions of 17. claim 13, wherein said C type ODN is ODN2395 or ODNM362 or ODN10101.

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